Non-azo naphthalimide dyes

ABSTRACT

A class of predominantly hydrophobic non-azo N-substituted 1,8-naphthalimide compounds, each bearing, at its 3-position, a nucleofuge and, at its 4-position, a heteroatomic electron-releasing group. The heteroatomic electron-releasing group is being characterized as having a heteroatom directly linked to the 4-position of the ring, and having at least one hydrogen directly attached to the heteroatom. Upon activation by an activating agent in an environment independent of the presence or absence of oxygen, these compounds generate activated species. The activated species initiate chemical changes in lipid bilayer membranes of viruses and other target cells. These changes can eradicate viruses and other target cells. The activated species can also cause structural changes in lipid and any associated proteins and polypeptides at a level beneath the surface of the membrane, leading to polymerization and crosslinking.

FIELD OF THE INVENTION

The present invention relates to predominantly hydrophobic non-azo1,8-naphthalimide dyes, their compositions, their preparations, andtheir uses. More specifically, the present invention relates topredominantly hydrophobic monomeric and dimeric non-azoN-substituted-1,8-naphthalimide dyes, their compositions, theirpreparations, and their uses.

Classical photodynamic therapy, which superficially resembles the newinvention described herein is a technique by which membrane alterationscan be made in a living cell and virus envelope by irradiation of a dyeincorporated into the membrane. See, U.S. Pat. Nos. 4,613,322;4,684,521; 4,649,151; and 4,878,891. This classical technique has beenproven to rely on the triplet sensitization of oxygen to form singletoxygen within the membrane. The hydroperoxides produced in the initialreaction between singlet oxygen and the unsaturated lipids of themembrane decompose to produce the observed membrane alterations, leadingto cell death or viral inactivation. Thus, the efficiency of thephotodynamic effect is directly related to the efficiency of theclassical dyes as a triplet sensitizer. Nearly all dyes currently usedfor the classical photodynamic therapy give good triplet yields uponirradiation. While selective alteration of either plasma ormitochondrial membranes can be achieved by using a dye with appropriatelocalization characteristics, the actual chemistry which produces themodification cannot be well controlled, and selective damage to themembrane cannot be affected.

There is currently no method available for selectively altering amembrane based upon selecting for one membrane over another on the basisof any simple membrane property, such as lipid composition, membranefluidity, surface proteins, integral proteins, or other similarfeatures. Moreover, all effective photochemical inactivators publishedto date rely on the production of singlet oxygen and the uncontrolledfree-radical chemistry of the hydroperoxides which it produces toachieve the cell kill or viral inactivation.

SUMMARY

According to the present invention, new non-azo 1,8-naphthalimide dyesor compounds are provided. These dyes can be "monomeric" or "dimeric."Further, their different uses are given. These new non-azo1,8-naphthalimide dyes are predominantly hydrophobic and, and afterbeing activated by a sufficient amount of activating agent in anenvironment independent of the presence or absence or oxygen, giveactivated derivatives or species. More specifically, the presentinvention relates to a "monomeric" non-azoN-substituted-1,8-naphthalimide compound bearing, at a 3-position, anucleofuge and, at a 4-position, a heteroatomic electron-releasinggroup, which is being characterized as having a heteroatom directlylinked to the 4-position and having at least one hydrogen directlyattached to the heteroatom. The present invention also relates to apredominantly hydrophobic "dimeric" non-azo bis-naphthalimide compound,having at least two 1,8-naphthalimide moieties each bearing, at a3-position, a nucleofuge and, at a 4-position, a heteroatomicelectron-releasing group which is being characterized as having aheteroatom directly linked to the 4-position and having at least onehydrogen directly attached to the heteroatom. The bis-naphthalimidecompound is further characterized as giving an activated derivativeafter being activated by a sufficient amount of activating agent in anenvironment independent of the presence or absence of oxygen. A non-azocompound or dye is one that does not possess a functional groupinghaving two nitrogen atoms connected by a double bond. A nucleofuge isany group which can be displaced from a molecule by a nucleophile.Examples of nucleofuge includes halogens, sulfonate esters, quartenaryammonium salts.

These new non-azo 1,8-naphthalimide dyes can be activated in thesimultaneous presence of an activating agent and the target tissue ororganism. Alternatively, these dyes can be pre-activated, in that theycan first be activated with an activating agent, and then introduced tothe target tissue or organism to accomplish their function in theabsence of the activating agent.

Exemplary uses of these dyes include:

Fluorescent probes. The unhalogenated naphthalimide dyes are highlyfluorescent lipophilic probes of low toxicity for the study of lipids inliving systems, including plasma membranes, organellar membranes,lipoprotein and atherosclerotic plaques without requiring either priorcovalent modification or fixation of the system being observed.

Protein immobilization. The immobilization of membrane-bound proteins,including surface antigens, glycoproteins, ionic channels, polypeptides,and enzymes in bilayers or micelles or both natural and syntheticlipids. The immobilization of surface antigens can occur in bilayersderived from a biological source. These immobilized species have thefollowing uses: elicitation of an immune response to the bound species;and development of new whole-virus, subunit, bacterial and cellvaccines.

Stabilization of lipid bilayers or micelles. These stabilized bilayersor micelles have the following potential uses: (i) applications todialysis; (ii) drug delivery, such as in stabilized liposomes; (iii)artificial semi-permeable membranes; (iv) biocompatible coatings, bothbiodegradable and non-biodegradable; (v) catalysis by bound species suchas enzymes; (vi) construction of stabilized membrane-bound receptors forsensing applications; and (vii) energy production by charge separationor generation of concentration gradients mediated by bound proteins,porphyrins, or other photoactivatable species.

Organism death and viral inactivation induced by an activating agent.The incorporation of these dyes into lipid bilayer membranes allows thefollowing uses: (i) sterilization of blood and blood products byselective incorporation into pathogenic organisms and light-inducedpathogen inactivation; (ii) sterilization of materials where thepresence of membrane-containing pathogens can be detrimental; and (iii)a new photochemical treatment, different from the classical photodynamictherapy, of certain cancers and tumors.

Encapsulation. Stabilized liposomes generated using these dyes tocross-link protein-containing liposomes can be used as encapsulatingagents whose resistance to heat, physical stress, pH, evaporation,lyophilization, detergents, freezing, high ionic strength solutions,agitation, organic solvents, lipases and proteases is much superior toavailable liposome technology. Potential uses of such encapsulatedmaterials include: (i) lyophilizable synthetic erythrocyte replacement(encapsulated hemoglobin); (ii) stabilized enzymes for organicsynthesis, including asymmetric synthesis of drugs in both aqueous andorganic solvent environments; (iii) time-release drug delivery where therate of delivery is determined by the concentration of the drug, thetime since administration, and the extent of cross-linking of themembrane; (iv) stabilized enzymes for therapeutic treatment of enzymedeficiency diseases; (v) stabilized encapsulated ion-selectivecomplexing agents such as EDTA (ethylenediamine tetracecetic acid) forthe treatment of diseases caused by an excess of a particular cation oranion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a to 1z, 1aa to 1zz, 1aaa to 1ccc show the structural formula ofCompounds I through LVI, respectively.

FIG. 2 is a diagram illustrating the general reaction schemes.

DETAILED DESCRIPTION OF THE INVENTION

The generic formula for "monomeric" non-azo 1,8-naphthalimide dye isrepresented by Compound I, having mixtures of stereoisomers, wherein:

R and R :

saturated straight-chain or branched-chain alkyl, C_(n) H_(2n+1)(n=1-30);

partially or totally fluorinated saturated straight-chain orbranched-chain alkyl, C_(n) H_(q) F_(2n-q+1) (n=1-30, 0≦q≦2n);

unsaturated straight-chain or branched-chain alkyl, C_(n) H_(2m+1)(n=1-30, 1≦m<n);

straight-chain or branched-chain alkyl, C_(n) H_(q) F_(2m-q+1) (n=1-30,1≦m<n, 0≦q≦2m);

alicyclic (monocyclic or polycyclic, fused-ring, bridged-ring orspirocyclic) alkyl, C_(n) H_(2m-1) (n=1-30, 1≦m≦n) with saturated orunsaturated side-chains (branched or unbranched);

partially or totally fluorinated alicyclic (monocyclic or polycyclic,fused-ring, bridged-ring or spirocyclic) alkyl, C_(n) H_(q) F_(2m+1)(n=1-30, 1≦m<n, 0≦q≦2m) with saturated or unsaturated side-chains(branched or unbranched);

aryl substituted branched, unbranched or alicyclic, saturated orunsaturated alkyl, ArC_(n) H_(2m+1) (n=1-30, 1≦m≦n), where Ar is anaromatic moiety (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole and their substituted and benzo derivatives) located eitherwithin the chain of the alkyl group or at a terminus;

partially or totally fluorinated aryl substituted branched, unbranchedor alicyclic, saturated or unsaturated alkyl, ArC_(n) H_(q) F_(2m-q+1)(n=1-30, 1≦m≦n, 0≦q≦2m), where Ar is an aromatic moiety (benzene,naphthalene, azulene, phenanthrene, anthracene, pyridine, quinoline,isoquinoline, purine, pyrimidine, pyrrole, indole, carbazole, furan,thiophene, imidazole, isoxazole, thiazole and their substituted andbenzo derivatives) located either within the chain of the alkyl group orat a terminus;

substituted saturated straight-chain or branched-chain alkyl, C_(n)H_(2n-p+1) Y_(p) (n=1-30, 1≦p≦10) where Y is a substituted aryl group(benzene, naphthalene, azulene, phenanthrene, anthracene, pyridine,quinoline, isoquinoline, purine, pyrimidine, pyrrole, indole, carbazole,furan, thiophene, imidazole, isoxazole, thiazole and their substitutedand benzo derivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, and ROPO₂²⁻), a silicon derivative (SiZ₃, where Z is alkyl, aryl, alkoxy,aryloxy, or halo), or a boron derivative (BZ₂, where Z is alkyl, aryl,alkoxy, aryloxy, or halo);

substituted partially or totally fluorinated straight-chain orbranched-chain alkyl, C_(n) H_(q) F_(2n-p-q+1) Y_(p) (n=1-30, 1≦p≦100≦q≦2n);

substituted unsaturated straight-chain or branched-chain alkyl, C_(n)H_(2m-p+1) Y_(p) (n=1-30, 1≦m<n, 1≦p≦10) where Y is a substituted arylgroup (benzene, naphthalene, azulene, phenanthrene, anthracene,pyridine, quinoline, isoquinoline, purine, pyrimidine, pyrrole, indole,carbazole, furan, thiophene, imidazole, isoxazole, thiazole and theirsubstituted and benzo derivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₃²⁻, and ROPO₂ ²⁻), a silicon derivative (SiZ₃, where Z is alkyl, aryl,alkoxy, aryloxy, or halo), or a boron derivative (BZ₂, where Z is alkyl,aryl, alkoxy, aryloxy, or halo);

substituted partially or totally fluorinated straight-chain orbranched-chain alkyl, C_(n) H_(q) F_(2m-p-q+1) Y_(p) (n=1-30, 1≦m<n,1≦p≦10, 0≦q≦2m);

substituted alicyclic (monocyclic or polycyclic, fused-ring,bridged-ring or spirocyclic) alkyl, C_(n) H_(2m+1) (n=1-30, 1≦m≦n) withsaturated or unsaturated side-chains (branched or unbranched) where Y isa substituted aryl group (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole and their substituted and benzo derivatives), a charged group(CO₂ ⁻, SO₃ ⁻, PO₂ ²⁻, and ROPO₂ ²⁻), a silicon derivative (SiZ₃, whereZ is alkyl, aryl, alkoxy, aryloxy, or halo), or a boron derivative (BZ₂,where Z is alkyl, aryl, alkoxy, aryloxy, or halo);

substituted partially or totally fluorinated alicyclic (monocyclic orpolycyclic, fused-ring, bridged-ring or spirocyclic) alkyl, C_(n) H_(q)F_(2m-p-q+1) Y_(p) (n=1-30, 1≦m<n, 1≦p≦10, 0≦q≦2m) with saturated orunsaturated side-chains (branched or unbranched) where Y is asubstituted aryl group (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole and their substituted and benzo derivatives), a charged group(CO₂ ⁻, SO₃ ⁻, PO₂ ²⁻, and ROPO₂ ²⁻), a silicon derivative (SiZ₃, whereZ is alkyl, aryl, alkoxy, aryloxy, or halo), or a boron derivative (BZ₂,where Z is alkyl, aryl, alkoxy, aryloxy, or halo);

substituted aryl substituted branched, unbranched or alicyclic,saturated or unsaturated alkyl, ArC_(n) H_(2m-p+1) Y_(p) (n=1-30, 1≦m≦n,1≦p≦10), where Ar is an aromatic moiety (benzene, naphthalene, azulene,phenanthrene, anthracene, pyridine, quinoline, isoquinoline, purine,pyrimidine, pyrrole, indole, carbazole, furan, thiophene, imidazole,isoxazole, thiazole and their substituted and benzo derivatives) locatedeither within the chain of the alkyl group or at a terminus, and where Yis a substituted aryl group (defined above), a charged group (CO₂ ⁻, SO₃⁻, PO₂ ²⁻, and ROPO₂ ²⁻), a silicon derivative (SiZ₃, where Z is alkyl,aryl, alkoxy, aryloxy, or halo), or a boron derivative (BZ₂, where Z isalkyl, aryl, alkoxy, aryloxy, or halo);

substituted partially or totally fluorinated aryl substituted branched,unbranched or alicyclic, saturated or unsaturated alkyl, ArC_(n) H_(q)F_(2m-p-q+1) Y_(p) (n=1-30, 1≦m≦n, 1≦p≦10, 0≦q≦2m), where Ar is anaromatic moiety (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole and their substituted and benzo derivatives) located eitherwithin the chain of the alkyl group or at a terminus, and where Y is asubstituted aryl group (defined above), a charged group (CO₂ ⁻, SO₃ ⁻,PO₂ ²⁻, and ROPO₂ ²⁻), a silicon derivative (SiZ₃, where Z is alkyl,aryl, alkoxy, aryloxy, or halo), or a boron derivative (BZ₂, where Z isalkyl, aryl, alkoxy, aryloxy, or halo);

a straight-chain or branched-chain group capable of complexing a metalion, C_(n) H_(2m+1) Y_(q) (n=1-30, 1≦q≦n, 1≦q≦n/2), where Y is O, S, Se,NH, N-R, N-Ar, PH, P-R and/or P-Ar and their acyl (including aminoacyland peptide) derivatives, and where R is saturated alkyl (C_(n)H_(2n+1), n=1-30), fluorinated saturated alkyl (C_(n) H_(q) F_(2n-q+1),n=1-30, 0≦q≦2n), unsaturated alkyl (C_(n) H_(2m+1), n=1-30, 1≦m<n),fluorinated unsaturated alkyl (C_(n) H_(q) F_(2m-q+1), n=1-30, 1≦m<n,0≦q≦2m), alicyclic (C_(n) H_(2m+1), n=1-30, 1≦m≦n), fluorinatedalicyclic (C_(n) H_(q) F_(2m-q+1), n=1-30, 1≦m<n, 0≦q≦2m), and where Aris an aromatic moiety (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole and their substituted and benzo derivatives) located eitherwithin the chain of the alkyl group or at a terminus;

a substituted alicyclic group capable of complexing a metal ion, C_(n)H_(2m+1) Y_(q) (n=1-30, 1≦q≦n, 1≦q≦n/2), where Y is O, S, Se, NH, N-R,N-Ar, PH, P-R and/or P-Ar and their acyl (including aminoacyl andpeptide) derivatives, and where R is saturated alkyl (C_(n) H_(2n+1),n=1-30), fluorinated saturated alkyl (C_(n) H_(q) F_(2n-q+1), n=1-30,0≦q≦2n), unsaturated alkyl (C_(n) H_(2m+1), n=1-30, 1≦m<n), fluorinatedunsaturated alkyl (C_(n) H_(q) F_(2m-q+1), n=1-30, 1≦m<n, 0≦q≦2m),alicyclic (C_(n) H_(2m+1), n=1-30, 1≦m≦n), fluorinated alicyclic (C_(n)H_(q) F_(2m-q+1), n=1-30, 1≦m<n, 0≦q≦2m), and where Ar is an aromaticmoiety (benzene, naphthalene, azulene, phenanthrene, anthracene,pyridine, quinoline, isoquinoline, purine, pyrimidine, pyrrole, indole,carbazole, furan, thiophene, imidazole, isoxazole, thiazole and theirsubstituted and benzo derivatives) located either within the chain ofthe alkyl group or at a terminus; or

a modified or unmodified biomolecule (steroids, phospholipids, mono-,di- and triglycerides, mono- and polysaccharides, nucleosides, andpolypeptides), where Y is a substituted aryl group (defined above), acharged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, and ROPO₂ ²⁻), a silicon derivative(SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, or halo), or a boronderivative (BZ₂, where Z is alkyl, aryl, alkoxy, aryloxy, or halo), or abiocompatible oligomer or polymer (polyglycolic acid);

halogen (F, Cl, Br, I);

sulfonate ester (alkanesulfonates, partially or totally fluorinatedalkanesulfonates, arenesulfonates, and partially or totally fluorinatedarenesulfonates); or

a nitrogen leaving group (diazonium ion);

or pharmaceutically acceptable salts thereof.

The generic formula for the "tail-to-tail dimeric" non-azo1,8-naphthalimide dye is represented by Compound II, having mixtures ofstereoisomers. The generic formula for the "head-to-head dimeric"non-azo 1,8-naphthalimide dye is represented by Compound III, havingmixtures of stereoisomers. Similarly, the generic formula for the"head-to-tail dimeric" non-azo 1,8-naphthalimide dye is represented byCompound IV, having mixtures of stereoisomers. The differentsubstituents and bridges for Compounds II, III and IV are:

R and R':

saturated straight-chain or branched-chain alkyl, C_(n) H_(2n+1)(n=1-30);

partially or totally fluorinated saturated straight-chain orbranched-chain alkyl, C_(n) H_(q) F_(2n-q+1) (n=1-30, 0≦q≦2n);

unsaturated straight-chain or branched-chain alkyl, C_(n) H_(2m+1)(n=1-30, 1≦m<n);

partially or totally fluorinated unsaturated straight-chain orbranched-chain alkyl, C_(n) H_(q) F_(2m-q+1) (n=1-30, 1≦m<n, 0≦q≦2m);

alicyclic (monocyclic or polycyclic, fused-ring, bridged-ring orspirocyclic) alkyl, C_(n) H_(2m+1) (n=1-30, 1≦m≦n) with saturated orunsaturated side-chains (branched or unbranched);

partially or totally fluorinated alicyclic (monocyclic or polycyclic,fused-ring, bridged-ring or spirocyclic) alkyl, C_(n) H_(q) F_(2m-q+1)(n=1-30, 1≦m<n, 0≦q≦2m) with saturated or unsaturated side-chains(branched or unbranched);

aryl substituted branched, unbranched or alicyclic, saturated orunsaturated alkyl, ArC_(n) H_(2m+1) (n=1-30, 1≦m≦n), where Ar is anaromatic moiety (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole and their substituted and benzo derivatives) located eitherwithin the chain of the alkyl group or at a terminus;

partially or totally fluorinated aryl substituted branched, unbranchedor alicyclic, saturated or unsaturated alkyl ArC_(n) H_(q) F_(2m-q+1)(n=1-30, 1≦m≦n, 0≦q≦2m), where Ar is an aromatic moiety (benzene,naphthalene, azulene, phenanthrene, anthracene, pyridine, quinoline,isoquinoline, purine, pyrimidine, pyrrole, indole, carbazole, furan,thiophene, imidazole, isoxazole, thiazole and their substituted andbenzo derivatives) located either within the chain of the alkyl group orat a terminus;

substituted saturated straight-chain or branched-chain alkyl, C_(n)H_(2n-p+1) Y_(p) (n=1-30, 1≦p≦10) where Y is a substituted aryl group(benzene, naphthalene, azulene, phenanthrene, anthracene, pyridine,quinoline, isoquinoline, purine, pyrimidine, pyrrole, indole, carbazole,furan, thiophene, imidazole, isoxazole, thiazole and their substitutedand benzo derivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, and ROPO₂²⁻), a silicon derivative (SiZ₃, where Z is alkyl, aryl, alkoxy,aryloxy, or halo), or a boron derivative (BZ₂, where Z is alkyl, aryl,alkoxy, aryloxy, or halo);

substituted partially or totally fluorinated straight-chain orbranched-chain alkyl, C_(n) H_(q) F_(2n-p-q+1) Y_(p) (n=1-30, 1≦p≦100≦q≦2n);

substituted unsaturated straight-chain or branched-chain alkyl, C_(n)H_(2m-p+1) Y_(p) (n=1-30, 1≦m<n, 1≦p≦10) where Y is a substituted arylgroup (benzene, naphthalene, azulene, phenanthrene, anthracene,pyridine, quinoline, isoquinoline, purine, pyrimidine, pyrrole, indole,carbazole, furan, thiophene, imidazole, isoxazole, thiazole and theirsubstituted and benzo derivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₂²⁻, and ROPO₂ ²⁻), a silicon derivative (SiZ₃, where Z is alkyl, aryl,alkoxy, aryloxy, or halo), or a boron derivative (BZ₂, where Z is alkyl,aryl, alkoxy, aryloxy, or halo);

substituted partially or totally fluorinated straight-chain orbranched-chain alkyl, C_(n) H_(q) F_(2m-p-q+1) Y_(p) (n=1-30, 1≦m<n,1≦p≦10, 0≦q≦2m);

substituted alicyclic (monocyclic or polycyclic, fused-ring,bridged-ring or spirocyclic) alkyl, C_(n) H_(2m+1) (n=1-30, 1≦m≦n) withsaturated or unsaturated side-chains (branched or unbranched) where Y isa substituted aryl group (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole and their substituted and benzo derivatives), a charged group(CO₂ ⁻, SO₃ ⁻, PO₂ ²⁻, and ROPO₂ ²⁻), a silicon derivative (SiZ₃, whereZ is alkyl, aryl, alkoxy, aryloxy, or halo), or a boron derivative (BZ₂,where Z is alkyl, aryl, alkoxy, aryloxy, or halo);

substituted partially or totally fluorinated alicyclic (monocyclic orpolycyclic, fused-ring, bridged-ring or spirocyclic) alkyl, C_(n) H_(q)F_(2m-p-q+1) Y_(p) (n=1-30, 1≦m<n, 1≦p≦10, 0≦q≦2m) with saturated orunsaturated side-chains (branched or unbranched) where Y is asubstituted aryl group (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole and their substituted and benzo derivatives), a charged group(CO₂ ⁻, SO₃ ⁻, PO₂ ²⁻, and ROPO₂ ²⁻), a silicon derivative (SiZ₃, whereZ is alkyl, aryl, alkoxy, aryloxy, or halo), or a boron derivative (BZ₂,where Z is alkyl, aryl, alkoxy, aryloxy, or halo);

substituted aryl substituted branched, unbranched or alicyclic,saturated or unsaturated alkyl, ArC_(n) H_(2m-p+1) Y_(p) (n=1-30, 1≦m≦n,1≦p≦10), where Ar is an aromatic moiety (benzene, naphthalene, azulene,phenanthrene, anthracene, pyridine, quinoline, isoquinoline, purine,pyrimidine, pyrrole, indole, carbazole, furan, thiophene, imidazole,isoxazole, thiazole and their substituted and benzo derivatives) locatedeither within the chain of the alkyl group or at a terminus, and where Yis a substituted (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻), a silicon derivative (SiZ₃,where Z is alkyl, aryl, alkoxy, aryloxy, or halo), or a boron derivative(BZ₂, where Z is alkyl, aryl, alkoxy, aryloxy, or halo);

substituted partially or totally fluorinated aryl substituted branched,unbranched or alicyclic, saturated or unsaturated alkyl, ArC_(n) H_(q)F_(2m-p-q+1) Y_(p) (n=1-30, 1≦m≦n, 1≦p≦10, 0≦q≦2m), where Ar is anaromatic moiety (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole and their substituted and benzo derivatives) located eitherwithin the chain of the alkyl group or at a terminus, and where Y is asubstituted aryl group (defined above), a charged group (CO₂ ⁻, SO₃ ⁻,PO₂ ²⁻, and ROPO₂ ²⁻), a silicon derivative (SiZ₃, where Z is alkyl,aryl, alkoxy, aryloxy, or halo), or a boron derivative (BZ₂, where Z isalkyl, aryl, alkoxy, aryloxy, or halo);

a straight-chain or branched-chain group capable of complexing a metalion, C_(n) H_(2m+1) Y_(q) (n=1-30, 1≦q≦n, 1≦q≦n/2), where Y is O, S, Se,NH, N-R, N-Ar, PH, P-R and/or P-Ar and their acyl (including aminoacyland peptide) derivatives, and where R is saturated alkyl (C_(n)H_(2n+1), n=1-30), fluorinated saturated alkyl (C_(n) H_(q) F_(2n-q+1),n=1-30, 0≦q≦2n), unsaturated alkyl (C_(n) H_(2m+1), n=1-30, 1≦m<n),fluorinated unsaturated alkyl (C_(n) H_(q) F_(2m-q+1), n=1-30, 1≦m<n,0≦q≦2m), alicyclic (C_(n) H_(2m+1), n=1-30, 1≦m≦n), fluorinatedalicyclic (C_(n) H_(q) F_(2m-q+1), n=1-30, 1≦m<n, 0≦q≦2m), and where Aris an aromatic moiety (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole and their substituted and benzo derivatives) located eitherwithin the chain of the alkyl group or at a terminus;

a substituted alicyclic group capable of complexing a metal ion, C_(n)H_(2m-+1) Y_(q) (n=1-30, 1≦q≦n, 1≦q≦n/2), where Y is O, S, Se, NH, N-R,N-Ar, PH, P-R and/or P-Ar and their acyl (including aminoacyl andpeptide) derivatives, and where R is saturated alkyl (C_(n) H_(2n-q+1),n=1-30), fluorinated saturated alkyl (C_(n) H_(q) F_(2n-q+1), n=1-30,0≦q≦2n), unsaturated alkyl (C_(n) H_(2m+1), n=1-30, 1≦m<n), fluorinatedunsaturated alkyl (C_(n) H_(q) F_(2m-q+1), n=1-30, 1≦m≦n, 0≦q≦2m),alicyclic (C_(n) H_(2m+1), n=1-30, 1≦m≦n), fluorinated alicyclic (C_(n)H_(q) F_(2m-q+1), n=1-30, 1≦m<n, 0≦q≦2m), and where Ar is an aromaticmoiety (benzene, naphthalene, azulene, phenanthrene, anthracene,pyridine, quinoline, isoquinoline, purine, pyrimidine, pyrrole, indole,carbazole, furan, thiophene, imidazole, isoxazole, thiazole and theirsubstituted and benzo derivatives) located either within the chain ofthe alkyl group or at a terminus; or

a modified or unmodified biomolecule (steroids, phospholipids, mono-,di- and triglycerides, mono- and polysaccharides, nucleosides, andpolypeptides), where Y is a substituted aryl group (defined above), acharged group (CO₂ ⁻, SO₃ ⁻, PO₂ ²⁻, and ROPO₂ ²⁻), a silicon derivative(SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, or halo), or a boronderivative (BZ₂, where Z is alkyl, aryl, alkoxy, aryloxy, or halo), or abiocompatible oligomer or polymer (polyglycolic acid);

X:

halogen (F, Cl, Br, I);

sulfonate ester (alkanesulfonates, partially or totally fluorinatedalkanesulfonates, arenesulfonates, and partially or totally fluorinatedarenesulfonates); or

a nitrogen leaving group (diazonium ion);

Q:

saturated straight-chain or branched-chain alkyl, C_(n) H₂ n (n=1-30);

partially or totally fluorinated saturated straight-chain orbranched-chain alkyl, C_(n) H_(q) F_(2n-q) (n=1-30, 0≦q≦2n);

unsaturated straight-chain or branched-chain alkyl, C_(n) H_(2m)(n=1-30, 1≦m<n);

partially or totally fluorinated unsaturated straight-chain orbranched-chain alkyl, C_(n) H_(q) F_(2m-q) (n=1-30, 1<m<n, 0≦q≦2m);alicyclic (monocyclic or polycyclic, fused-ring, bridged-ring orspirocyclic) alkyl, C_(n) H_(2m) (n=1-30, 1≦m≦n) with saturated orunsaturated side-chains (branched or unbranched);

partially or totally fluorinated alicyclic (monocyclic or polycyclic,fused-ring, bridged-ring or spirocyclic) alkyl, C_(n) H_(q) F_(2m-q)(n=1-30, 1≦m≦n, 0≦q≦2m) with saturated or unsaturated side-chains(branched or unbranched);

aryl substituted branched, unbranched or alicyclic, saturated orunsaturated alkyl, ArC_(n) H_(2m) (n=1-30, 1≦m≦n), where Ar is anaromatic moiety (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole and their substituted and benzo derivatives) located eitherwithin the chain of the alkyl group or at a terminus;

partially or totally fluorinated aryl substituted branched, unbranchedor alicyclic, saturated or unsaturated alkyl, ArC_(n) H_(q) F_(2m-q)(n=1-30, 1m≦n, 0≦q≦2m), where Ar is an aromatic moiety (benzene,naphthalene, azulene, phenanthrene, anthracene, pyridine, quinoline,isoquinoline, purine, pyrimidine, pyrrole, indole, carbazole, furan,thiophene, imidazole, isoxazole, thiazole and their substituted andbenzo derivatives) located either within the chain of the alkyl group orat a terminus;

substituted saturated straight-chain or branched-chain alkyl, C_(n)H_(2n-p) Y_(p) (n=1-30, 1≦p≦10) where Y is a substituted aryl group(benzene, naphthalene, azulene, phenanthrene, anthracene, pyridine,quinoline, isoquinoline, purine, pyrimidine, pyrrole, indole, carbazole,furan, thiophene, imidazole, isoxazole, thiazole and their substitutedand benzo derivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₂ ²⁻, and ROPO₂²⁻), a silicon derivative (SiZ₃, where Z is alkyl, aryl, alkoxy,aryloxy, or halo), or a boron derivative (BZ₂, where Z is alkyl, aryl,alkoxy, aryloxy, or halo);

substituted partially or totally fluorinated straight-chain orbranched-chain alkyl, C_(n) H_(q) F_(2n-p-q) Y_(p) (n=1-30, 1≦p≦100≦q≦2n);

substituted unsaturated straight-chain or branched-chain alkyl, C_(n)H_(2m-p) Y_(p) (n=1-30, 1≦m≦n, 1≦p≦10) where Y is a substituted arylgroup (benzene, naphthalene, azulene, phenanthrene, anthracene,pyridine, quinoline, isoquinoline, purine, pyrimidine, pyrrole, indole,carbazole, furan, thiophene, imidazole, isoxazole, thiazole and theirsubstituted and benzo derivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₂²⁻, and ROPO₂ ²⁻), a silicon derivative (SiZ₃, where Z is alkyl, aryl,alkoxy, aryloxy, or halo), or a boron derivative (BZ₂, where Z is alkyl,aryl, alkoxy, aryloxy, or halo);

substituted partially or totally fluorinated straight-chain orbranched-chain alkyl, C_(n) H_(q) F_(2m-p-q) Y_(p) (n=1-30, 1≦m<n,1≦p≦10, 0≦q≦2m);

substituted alicyclic (monocyclic or polycyclic, fused-ring,bridged-ring or spirocyclic) alkyl, C_(n) H_(2m) (n=1-30, 1≦m≦n) withsaturated or unsaturated side-chains (branched or unbranched) where Y isa substituted aryl group (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole and their substituted and benzo derivatives), a charged group(CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, ), a silicon derivative (SiZ₃, where Z is alkyl,aryl, alkoxy, aryloxy, or halo), or a boron derivative (BZ₂, where Z isalkyl, aryl, alkoxy, aryloxy, or halo);

substituted partially or totally fluorinated alicyclic (monocyclic orpolycyclic, fused-ring, bridged-ring or spirocyclic) alkyl, C_(n) H_(q)F_(2m-p-q) Y_(p) (n=1-30, 1≦m<n, 1≦p≦10, 0≦q≦2m) with saturated orunsaturated side-chains (branched or unbranched) where Y is asubstituted aryl group (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole and their substituted and benzo derivatives), a charged group(CO₂ ⁻, SO₃ ⁻, PO₂ ²⁻, and ROPO₂ ²⁻), a silicon derivative (SiZ₃, whereZ is alkyl, aryl, alkoxy, aryloxy, or halo), or a boron derivative (BZ₂,where Z is alkyl, aryl, alkoxy, aryloxy, or halo);

substituted aryl substituted branched, unbranched or alicyclic,saturated or unsaturated alkyl, ArC_(n) H_(2m-p) Y_(p) (n=1-30, 1≦m≦n,1<p≦10), where Ar is an aromatic moiety (benzene, naphthalene, azulene,phenanthrene, anthracene, pyridine, quinoline, isoquinoline, purine,pyrimidine, pyrrole, indole, carbazole, furan, thiophene, imidazole,isoxazole, thiazole and their substituted and benzo derivatives) locatedeither within the chain of the alkyl group or at a terminus, and where Yis a substituted aryl group (defined above), a charged group (CO₂ ⁻, SO₃⁻, PO₂ ²⁻, and ROPO₂ ²⁻), a silicon derivative (SiZ₃, where Z is alkyl,aryl, alkoxy, aryloxy, or halo), or a boron derivative (BZ₂, where Z isalkyl, aryl, alkoxy, aryloxy, or halo);

substituted partially or totally fluorinated aryl substituted branched,unbranched or alicyclic, saturated or unsaturated alkyl, ArC_(n) H_(q)F_(2m-p-q) Y_(p) (n=1-30, 1≦m≦n, 1≦p≦10, 0≦q≦2m), where Ar is anaromatic moiety (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole and their substituted and benzo derivatives) located eitherwithin the chain of the alkyl group or at a terminus, and where Y is asubstituted aryl group (defined above), a charged group (CO₂ ⁻, SO₃ ⁻,PO₃ ²⁻, and ROPO₂ ²⁻), a silicon derivative (SiZ₃, where Z is alkyl,aryl, alkoxy, aryloxy, or halo), or a boron derivative (BZ₂, where Z isalkyl, aryl, alkoxy, aryloxy, or halo);

a straight-chain or branched-chain group capable of complexing a metalion, C_(n) H_(2m) Y_(q) (n=1-30, 1≦q≦n, 1≦q≦n/2), where Y is O, S, Se,NH, N-R, N-Ar, PH, P-R and/or P-Ar and their acyl (including aminoacyland peptide) derivatives, and where R is saturated alkyl (C_(n) H_(2n),n=1-30), fluorinated saturated alkyl (C_(n) H_(q) F_(2n-q), n=1-30,0≦q≦2n), unsaturated alkyl (C_(n) H_(2m), n=1-30, 1≦m<n), fluorinatedunsaturated alkyl (C_(n) H_(q) F_(2m-q), n=1-30, 1≦m<n, 0≦q≦2m),alicyclic (C_(n) H_(2m), n=1-30, 1≦m≦n), fluorinated alicyclic (C_(n)H_(q) F_(2m-q), n=1-30, 1≦m<n, 0≦q≦2m), and where Ar is an aromaticmoiety (benzene, naphthalene, azulene, phenanthrene, anthracene,pyridine, quinoline, isoquinoline, purine, pyrimidine, pyrrole, indole,carbazole, furan, thiophene, imidazole, isoxazole, thiazole and theirsubstituted and benzo derivatives) located either within the chain ofthe alkyl group or at a terminus;

a substituted alicyclic group capable of complexing a metal ion, C_(n)H_(2m) Y_(q) (n=1-30, 1≦q≦n, 1≦q≦n/2), where Y is O, S, Se, NH, N-R,N-Ar, PH, P-R and/or P-Ar and their acyl (including aminoacyl andpeptide) derivatives, and where R is saturated alkyl (C_(n) H_(2n),n=1-30), fluorinated saturated alkyl (C_(n) H_(q) F_(2n-q), n=1-30,0≦q≦2n), unsaturated alkyl (C_(n) H_(2m), n=1-30, 1≦m<n), fluorinatedunsaturated alkyl (C_(n) H_(q) F_(2m-q), n=1-30, 1≦m≦n, 0≦q≦2m),alicyclic (C_(n) H_(2m), n=1-30, 1≦m≦n), fluorinated alicyclic (C_(n)H_(q) F_(2m-q), n=1-30, 1≦m<n, 0≦q≦2m), and where Ar is an aromaticmoiety (benzene, naphthalene, azulene, phenanthrene, anthracene,pyridine, quinoline, isoquinoline, purine, pyrimidine, pyrrole, indole,carbazole, furan, thiophene, imidazole, isoxazole, thiazole and theirsubstituted and benzo derivatives) located either within the chain ofthe alkyl group or at a terminus; or

a modified or unmodified biomolecule (steroids, phospholipids, mono-,di- and triglycerides, mono- and polysaccharides, nucleosides, andpolypeptides), where Y is a substituted aryl group (defined above), acharged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, and ROPO₂ ²⁻), a silicon derivative(SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, or halo), or a boronderivative (BZ₂, where Z is alkyl, aryl, alkoxy, aryloxy, or halo), or abiocompatible oligomer or polymer (polyglycolic acid); and

pharmaceutically acceptable salts thereof.

CHEMICAL SYNTHESES

FIG. 2 illustrates some of the general reaction schemes.

I. General Procedure for Synthesis of4-Alkylamino-N-alkyl-1,8-naphthalimides with two different alkyl groups.(I, X=H; XV, XVI, XXI, XXII, XXV, XXVI, XXVII, XXVIII, XXXIII, XXXV,XVII, XXVIII, XXIX, XL, XLI).

(a) Synthesis of the 4-chloro-N-alkyl-1,8-naphthalimide.

Recrystallized 4-chloro-1,8-naphthalic anhydride (1 equivalent) issuspended in toluene or 1,2-dimethoxyethane (20-25 mL/g) and the primaryamine (1-2 equivalent) is added. The mixture is heated to reflux,whereupon the color of the reaction mixture gradually changes fromyellow to off-white. After 24 hours, the reaction mixture is cooled, andthe solvent is removed by evaporation under reduced pressure. The solidresidue of the 4-chloro-N-alkyl-1,8-naphthalimide is recrystallized frommethanol.

Specific example:

Recrystallized 4-chloro-1,8-naphthalic anhydride (5.814 g, 25.0 mmol)was suspended in toluene (120 mL) and 1-hexylamine (3.30 mL, 25.0 mmol)was added by graduated pipette. The mixture was heated to reflux, andthe color of the solution gradually changed from yellow to off-white.After 24 hours, the solids had all dissolved, and the solution wasallowed to cool. The solvent was removed by evaporation under reducedpressure, and the solid residue was recrystallized from methanol toafford 4-chloro-N-hexyl-1,8-naphthalimide (LV, R=n-C₆ H₁₃, 7.259 g, 92%)as an off-white solid.

Representative compounds prepared by this method:

    ______________________________________                                                 mass of    amount                                                    R        anhydride  of amine  solvent yield                                   ______________________________________                                        n-C.sub.4 H.sub.9                                                                      4.185 g    1.80   mL   toluene 96%                                   n-C.sub.6 H.sub.13                                                                     5.499 g    3.10   mL   toluene 90%                                   n-C.sub.8 H.sub.17                                                                     4.124 g    2.90   mL   toluene 91%                                   n-C.sub.10 H.sub.21                                                                    2.525 g    2.0    mL   toluene 70%                                   n-C.sub.14 H.sub.29                                                                    0.536 g    0.656  g    toluene  95%*                                 n-C.sub.16 H.sub.33                                                                    1.018 g    1.071  g    toluene 44%                                   n-C.sub.18 H.sub.35                                                                    1.016 g    1.480  g    toluene 53%                                   C.sub.6 H.sub.5                                                                        1.028 g    42     mL   PhNH.sub.2                                                                            70%                                   p-BrC.sub.6 H.sub.4                                                                    1.002 g    1.486  g    n-BuOH  34%                                   ______________________________________                                         *Crude yield                                                             

(b) Synthesis of the 4-alkylamino-N-alkyl-1,8-naphthalimide.

The 4-chloro-N-alkyl-1,8-naphthalimide (1 equivalent is dissolved orsuspended in the primary amine (2-20 mL/g) and the solution is heated toreflux for 18-24 hours. The resultant solution is allowed to cool, andthe solvent is removed by evaporation under reduced pressure. The solidthus obtained is recrystallized from methanol. Yields are difficult todetermine because of the propensity of many of these compounds to formsolvates.

Specific example:

A stirred solution of 4-chloro-N-hexyl-1,8-naphthalimide (LV, R=n-C₆ H₁₃; 10.12 g, 32 mmol) in ethylenediamine (25 mL) was heated under refluxfor 18 hours. After this time, the reaction mixture was cooled to roomtemperature. The solution solidified, and the excess ethylenediamine wasremoved by evaporation under reduced pressure. The residual yellow solidwas recrystallized from methanol to afford the product as yellowcrystals (12.56 g).

Representative compounds prepared by this method:

    ______________________________________                                                            mass      volume                                          R       R'          of imide  of amine                                                                             yield                                    ______________________________________                                        n-C.sub.4 H.sub.9                                                                     CH.sub.2 CH.sub.2 NH.sub.2                                                                0.78 g    16 mL  0.80 g                                   n-C.sub.6 H.sub.13                                                                    CH.sub.2 CH.sub.2 NH.sub.2                                                                1.62 g    25 mL  1.98 g                                   n-C.sub.8 H.sub.17                                                                    CH.sub.2 CH.sub.2 NH.sub.2                                                                1.09 g    40 mL  1.06 g                                   n-C.sub.14 H.sub.29                                                                   CH.sub.2 CH.sub.2 NH.sub.2                                                                1.113 g   11 mL  0.930 g                                  n-C.sub.16 H.sub.33                                                                   CH.sub.2 CH.sub.2 NH.sub.2                                                                0.888 g   12 mL  75%*                                     n-C.sub.18 H.sub.35                                                                   CH.sub.2 CH.sub.2 NH.sub.2                                                                0.62 g    11.5 mL                                                                              0.60 g                                   n-C.sub.6 H.sub.13                                                                    CH.sub.2 CH.sub.2 OH                                                                      1.502 g   50 mL  1.531 g                                  ______________________________________                                         *Based on unsolvated formula.                                            

II. General Procedure for Synthesis of4-Alkylamino-N-alkyl-1,8-naphthalimides with two identical alkyl groups.(I, X=H, V, VI, IX, X).

A stirred suspension of 4-chloro-1,8-naphthalic anhydride (1 equivalent)in this primary amine (10-30 mL/g) is heated under reflux for 18-24hours. The resultant solution is allowed to cool to room temperature,and the amine is then removed by evaporation under reduced pressure. Thecrude product, which is usually an oily solid, is recrystallized frommethanol.

Specific example:

Freshly recrystallized 4-chloro-1,8-naphthalic anhydride (4.32 g, 18.5mmol) was dissolved in 1-aminohexane (120 mL), and the resultantsolution was heated under reflux for 18 hours. The red solution wasallowed to cool, and the 1-aminohexane was removed under reducedpressure to afford the product as an oily solid. Recrystallization frommethanol afforded 4-(hexyl)amino-N-hexyl-1,8-naphthalimide (I, R=R'-n-C₆H₁₃ ; 5.72 g, 81%).

Representative compounds prepared by this method:

    ______________________________________                                                 mass of    amount                                                    R and R' anhydride  of amine yield   (solvent)                                ______________________________________                                        n-C.sub.4 H.sub.9                                                                      3.106 g    25 mL    55%     --                                       s-C.sub.4 H.sub.9                                                                      2.511 g    100 mL   79%     --                                       i-C.sub.4 H.sub.9                                                                      2.298 g    44 mL    67%     --                                       n-C.sub.6 H.sub.13                                                                     5.003 g    100 mL   61-83%  --                                       n-C.sub.8 H.sub.17                                                                     5.101 g    56 mL    24%     --                                       n-C.sub.9 H.sub.19                                                                     1.017 g    0.8 mL   51%     (DMF)                                    CH.sub.2 CH.sub.2 NH.sub.2                                                             5.100 g    100 mL   31%     --                                       CH.sub.2 CH.sub.2 OH                                                                   3.106 g    25 mL    52%     --                                       ______________________________________                                    

III. General procedure for bromination:

The naphthalimide is dissolved in a minimum volume of carbontetrachloride (typically 20-40 mL/g) and a slight (10 mol %) excess ofbromine is added. The reaction is stirred at ambient temperature, and ismonitored by thin layer chromatography. When all of the startingmaterial has been consumed, the solvent and the excess bromine areremoved by evaporation under reduced pressure. The crude product isrecrystallized from methanol to afford the3-bromo-4-(alkyl)amino-N-alkyl-1,8-naphthalimide (I, X=Br). Ifnecessary, chromatographic purification can be carried out by columnchromatography on alumina (200 g adsorbent per g of compound) elutingwith chloroform-hexane mixtures.

Specific example:

To a solution of 4-(hexyl)amino-N-hexyl-1,8-naphthalimide (0.531 g, 1.40mmol) in carbon tetrachloride (20 mL) was added bromine (0.08 mL, 1.5mmol) by graduated pipette. The reaction mixture was allowed to stir atroom temperature for 3.5 hours, and the solvent was then removed byevaporation under reduced pressure. The resultant yellow solid wasrecrystallized from methanol to afford3-bromo-4-(hexyl)amino-N-hexyl-1,8-naphthalimide (VII, 0.435 g, 67%) asbright yellow needles.

Representative compounds prepared by this method:

    ______________________________________                                                               mass                                                   R          R'          of imide yield                                         ______________________________________                                        n-C.sub.4 H.sub.9                                                                        n-C.sub.4 H.sub.9                                                                         0.407 9  0.438 g                                       n-C.sub.6 H.sub.13                                                                       n-C.sub.6 H.sub.13                                                                        0.531 g  0.435 g                                       n-C.sub.8 H.sub.17                                                                       n-C.sub.8 H.sub.17                                                                        0.584 g  0.584 g                                       n-C.sub.6 H.sub.13                                                                       CH.sub.2 CH.sub.2 NH.sub.2                                                                0.819 g    0.1 g*                                      ______________________________________                                         *Yields were low; these compounds must be prepared and purified under         lowlight or redlight conditions.                                         

EVALUATION

This invention pertains to a class of predominantly hydrophobic non-azo1,8-naphthalimide dyes whose biological and chemical activity ispresumably due to a new mechanism of action termedphototautomerization-alkylation which probably proceeds in three primarysteps. Step one is activation by an activating agent to generate anenergetically excited species. In step two, the initially excitedmolecular species undergoes tautomerization to generate analpha-haloimine which is also a gamma-halocrotonamide species. Such aspecies is a highly reactive alkylating agent. In step three, the activespecies reacts covalently with available substantially nucleophilicgroups available on biological molecules such as cysteinyl, cystinyl,tryptophanyl, tyrosyl, seryl amino acid residues of peptides andproteins. Unlike many other photoactive dyes, these predominantlyhydrophobic non-azo 1,8-naphthalimide dyes can be activated in anenvironment independent of the presence or absence of oxygen. They donot rely on the production of singlet oxygen for their mechanism ofaction.

This class of non-azo 1,8-naphthalimide dyes in the absence of asuitable activating agent, such as electromagnetic radiation, issubstantially nontoxic to viruses, bacteria, protozoans or to otherbiological cells and tissues. These new dyes are readily incorporatedinto lipid bilayers, membranes and micelles of natural or syntheticorigin. Due to the sequestration of these lipophilic dyes intohydrophobic regions of proteins, viruses, both cellular and liposomalmembranes, and tissues having a high density of biomolecularnucleophilic groups, the covalent reactions initiated by the activatedform of these dyes can result in chemical alteration of amino acidresidues, of protein and peptide conformation and function, and cancross-link the amino acid residues, peptides, and proteins. Thus, thisclass of dyes can be used to kill or inactivate viral, bacterial, andprotozoal infective agents, neoplastic and cancerous cells, to linkdesired molecular and biomolecular species to peptides, proteins, cells,and biological tissues as well as other substrates containingnucleophilic groups, and to cross-link peptides, proteins, tissues, andother substrates containing nucleophilic groups selectively uponapplication of an activating agent, such as electromagnetic radiationwith wavelength corresponding in absorption spectrum of the dyeabsorption spectrum. The appropriate electromagnetic radiationabsorption spectrum includes the ultraviolet through visible light tonear infrared and the K-alpha, etc., X-ray absorption energies of themolecular halogen substituent. Other activating agents include thermalneutrons which could be used to activate boron-containing1,8-naphthalimide dyes.

The partitioning of these non-azo 1,8-naphthalimide dyes intohydrophobic regions such as the interior of the lipid bilayer ofliposomes and the capability of activating covalent chemical reactionswith nucleophilic amino acid residues allows cross-linking of theintramembrane regions of peptides and proteins associated with thebilayer membrane selectively upon exposure to light. No cross-linkingoccurs until being activated by an activating agent, such as light. Thisis in contrast to the use of conventional dark-acting chemicalcross-linking or linking agents such as formaldehyde, gluteraldehyde,succinimidyl esters, iodoactamides, or maleimides which act immediatelyupon contact with the appropriate protein residues during mixing byphysical agitation or diffusion.

Thus, with the use of conventional dark-acting agents, it is difficultto delay initiation of the cross-linking chemical reaction until achosen time during appropriate mixing or sequestration of mixturecomponents. The use of the light activatable 1,8-naphthalimide dyesallows temporary delay of cross-linking until desired mixing orsequestration of liposomal membrane constituents and internalizedcomponents has been accomplished. This capability allows, for example,synthesis of liposomes containing a completely cross-linked network ofGramacidin-D peptide units within the lipid bilayer and which contain apeptide or protein or other molecular species within the interior suchas hemoglobin. The structural and functional integrity of thesemolecules has been maintained during the cross-linking process becausethe lipophilic cross-linking dye is physically isolated from thehydrophilic interior and incapable of chemical reaction with theinternalized molecular species. Additionally, the lack of cross-linkingin the dark by 1,8-naphthalimide dyes allows greater facility of mixingof the constituent in the dark without fear of reaction.

Light induced covalent linking of the non-azo 1,8-naphthalimide dyeswith a substrate also allows their use in linking a desired biomolecularor pharmaceutical agent to target biological cells or to the surface ofa biological tissue at a desired time.

Use of light activation of these dyes also allows cross-linking ofproteins at different tissue surfaces in order to bond or weldbiological tissues together and avoid unwanted intermediate reactions.The advantage of light induced tissue welding over thermally inducedwelding is less damage to tissue adjacent to the welded area due to lackof heating of surrounding tissues by diffusion during the procedure.

Strong localization of the lipophilic non-azo 1,8-naphthalimide dyeswithin the hydrophobic regions of enveloped or naked virus particlesallows efficient killing or inactivation of the infective capability ofthe virus. Delay of the biological action of the dyes until activationallows temporal control during the process.

Localization of the light activated covalent reactions of the dyes withnucleophilic amino residues allows cross-linking of viral proteinsbetween their hydrophobic regions, well away from the hydrophilicregions of these proteins which are recognized as antigenic in thegeneration of infective host antibodies and immunity. Thus, they arehighly effective for making damaged, altered, or killed whole virusparticle vaccines while preserving the outer hydrophilic exposed viralsurface shapes which are important in antibody generation, andimmunogenicity. Similarly, these predominantly hydrophobic non-azo1,8-naphthalimide dyes are efficient in making damaged, altered orkilled whole bacterial vaccine while preserving the immunogenicity.

Because these lipophilic dyes are both taken up by cancer cells, theyoffer an excellent vehicle for loading of a Boron isotope-containingcompound into neoplastic cells for tumor treatment via activation withthermal neutrons.

As used herein, the word "dye" is interchangeable with the word"compound", as referred to non-azo 1,8-naphthalimides.

The term "pre-activated" as used herein denotes that the1,8-naphthalimide dye is activated, sensitized, or excited outside theanimal or human body, or outside the body tissue. Thus, the term"pre-activated" denotes that the activation of the dye is accomplishedaway from the body tissue to be treated, away from the target tumorcells or target biological pathogenic contaminants. Hence, theactivation step in the "pre-activated" method is carried out before, notafter, the dye has interacted with the target tumor cells or with otherpathogenic biological contaminants. In fact, the activation step in the"pre-activated" method is carried out prior to the dye being broughtinto contact with the tissue to be treated. There is no requirement forfurther activation at the target sites once the therapeutic agent hasbeen pre-activated. The pre-activated therapeutic agent so generated hasa measurable and clinically useful shelf life time.

The activating agent as used herein denotes a means or an agent that iscapable of activating, exciting, or sensitizing a photoactive compound.The activating agent can be radiated energy, electromagnetic energy,laser, electric current, electrons, thermal neutrons or chemicals. Theelectromagnetic spectrum can include visible light, xenon light, laserlight, near infrared and ultraviolet light. The laser or other radiationenergy can be continuous or pulsed. The pulsed energy used is such thatthe energy supplied has a multiple number of short pulses of relativelyhigh energy, but at the same time, has a much lower average energy rate.The laser could be gold vapor laser, xenon arc lamp laser, argon laser,a crystal laser, a gas discharge laser, an excimer laser, krypton laser,argon ion pumped dye laser, or hollow cathode metal vapor laser, andothers. Even sources such as conventional filament lamp source withappropriate filtering, an arc lamp source with appropriate filtering, oreven a pulsed xenon flash lamp with appropriate filtering could be used.

The term "interact" as used herein denotes the general phenomena ofhaving the therapeutic agent adhere to, accumulate in, or associatedwith the tumor cells or other pathogenic biological contaminantsinfecting a body tissue.

The term "pathogenic biological contaminants" is to be understood toinclude: viruses, enveloped or not enveloped; microorganisms; parasites;bacteria and the like.

"Tumors" or "tumor cells" is understood to encompass malignant andnon-malignant types and include, among others: cancer of the bone andconnective tissues; cancer of the eyes; leukemias; lymphomas; myelomas;melanomas, breast cancer, lung cancer, ovarian cancer as well as othertypes of cancer and solid tumors.

The term "body tissue" as used herein is to be understood to include"body fluid," red blood cells, white blood cells, platelets, cryoprecipitate from blood plasma, other plasma proteins, bone marrow, skin,cornea, ligament, tendon and other tissues from an animal or a human.

The term "body fluid" as used herein is to be understood to includewhole blood, any formed elements of the blood, blood plasma, serum,fluids containing such components, fluids from plasmapheresis, plasmafibrinogen, cryo-poor plasma, albumin, gamma globulins, semen, and otherfluids introduced or intravenously injected into the body of a patientor an animal using known administration techniques. The term "bodyfluid" is to be understood to include body fluid prior to, or after,physical as well as chemical fractionation, separation or freezing.

The term "external" as used herein is to denote outside the animal orhuman body.

The term "animal" as used herein is to denote any warm-blooded animal;this includes human and other domestic and farm animals.

The term "carrier" as used herein denotes a vehicle, a solutioncontaining water, buffers, serum, serum proteins, lipoproteins,artificial bio-membranes, micelles, liposomes, monoclonal antibodies,carbohydrates, cyclodextrans, organic solvents or other pharmaceuticallyacceptable, or compatible, solutions. The carrier, or vehicle, used ispharmaceutically compatible in that it is relatively non-toxic to thenormal cells and normal tissues and it does not react with the solute ortherapeutic agent contained therein.

The phrase "effective amount" as used herein is to denote theconcentration or level of the therapeutic agent that can attain aparticular end, such as cross-linking, a cure or a destruction of theundesirable cells, such as tumor cells, or pathogenic biologicalcontaminants, without producing pronounced toxic symptoms.

SYNTHETIC MEMBRANE SYSTEMS

The behavior of the dyes of the present invention has been studied insynthetic vesicles, prepared by standard protocols of ethanol injectionor sonication, and micelles for two major reasons: Firstly, the vesiclesystem is easier to control and simpler to understand than a biologicalsystem with its full complement of proteins and its full range of lipidcomponents. Secondly, many of the potential application discussed aboveare based upon lipid bilayer or micelle technology. The descriptionsbelow refer to explicit cases to aid in evaluation of the invention.

(1) Binding Kinetics.

All of the vehicles used in these studies were prepared to have a finallipid, egg lecithin+cholesterol concentration of 10 mg/ml. Cholesterolconcentrations were varied between 0 and 45 mol % by varying the lipidcomposition from which the vesicles were produced. The subject dyes wereadded in 10% aqueous isopropyl alcohol solutions to vesicle suspensionsin water. The dyes are non-fluorescent in 10% aqueous isopropy alcohol,but are highly fluorescent in vesicles. The increase in fluorescence wasmonitored at an excitation wavelength of 460 nm and an emissionwavelength of 519 nm. Fluorescence intensity was measured as a functionof time, and fit to a standard first-order kinetic plot. Compound 1b isCompound I, wherein R=R¹ =n-hexyl and X=H. The rate constants are givenin Table 1.

                  TABLE 1                                                         ______________________________________                                        Rate Constants for Binding of Compound VII to                                 Synthetic Vesicles of β-Oleyl-γ-stearoylphospahtidyl Choline       mol % cholesterol                                                                            rate constant (s.sup.-1)                                       ______________________________________                                         0             0.814 ± 0.005 × 10.sup.-3                             15             1.020 ± 0.005 × 10.sup.-3                             30              1.54 ± 0.02 × 10.sup.-3                              45              2.75 ± 0.01 × 10.sup.-3                              ______________________________________                                    

(2) Bleaching Kinetics.

The dye was incorporated into synthetic vesicles as described above. Thevesicles were irradiated with a 150-W Oriel xenon arc lamp at a distanceof 4 cm from the lens at a thermostated temperature of 20° C. The changein fluorescence intensity with time was determined above, and theresults plotted as a standard first order plot. The results are given inTable 2.

                  TABLE 2                                                         ______________________________________                                        Rate Constants for Bleaching of Compound VII in                               Synthetic Vesicles of β-Oleyl-γ-stearoylphospahtidyl Choline       mol % cholesterol                                                                            rate constant (s.sup.-1)                                       ______________________________________                                         0             1.08 ± 0.08 × 10.sup.-3                               15             2.00 ± 0.13 × 10.sup.-3                               30             3.08 ± 0.20 × 10.sup.-3                               45              3.1 ± 0.9 × 10.sup.-3                                ______________________________________                                    

Synthetic vesicles of β,γ-distearoylphosphatidyl choline containingCompound VII do not bleach, and show only very slow photodegradation ofthe dye.

(3) Effect of Irradiation on Lipid Structure.

Upon irradiation, crude phospholipid vesicles containing Compound VIIgive rise to new lipids whose t.l.c. mobility is lower than the startingphospholipid mixture, indicating probable cross-linking.

(4) Effects of Irradiation on Membrane-Bound Protein Function.

Cytochrome C, a mitochondrial protein which is peripherally associatedwith membranes was studied as a model for the effect ofphotomodification of the membrane lipids on protein function. At pH 7.5and 20° C., the rate of oxidation of vesicle-bound cytochrome c (7×10⁻⁶M) by CO(phen)3³ + was 25±1 s⁻¹. Upon addition of Compound VII (1×10⁻⁷M) to the vesicles containing reduced cytochrome c, and subsequentirradiation as described above, the rate constant for oxidation of thebound cytochrome c under the same conditions was reduced to 2±1 s⁻¹.

BIOLOGICAL STUDIES

The behavior of the subject dyes in living systems has been exploredusing a variety of cells and media, as well as viruses. Binding andlight-kill studies have been carried out using H9 cells, a transformedimmortalized T-lymphoma, and Daudi cells as representative mammaliancells.

(1) Uptake Into Living Cells.

The staining of cells with Compound VII or Compound 1b was effectedusing the following protocol: H9 cells were counted using ahemocytometer. 1.25×10⁷ cells at 600×g. The pellet was resuspended in 25ml of fresh, prewarmed RPMI-1640+10% FCS (this gives 5×10⁵ cells/ml).The suspension was divided into 3 aliquots of 7 ml each, and Compound 1b(5 mg/ml in 2-propanol) was added to give solutions of 1, 5 and 25 μM.The solutions were maintained at a constant temperature, and samples (1ml) were taken at time 0, 0.5, 1, 2, 3, and 7.5 hours, and centrifugedat 500×g for 30 s, resuspended in 1 ml fresh RPMI-1640+10% FCS, andcentrifuged again. For measurement, the pellet was diluted in 3 mlPBS+0.1% CTAB immediately prior to fluorescence measurement.Fluorescence measurements were made using an excitation wavelength of451 nm, and an emission wavelength of 526 nm. The results are plotted asa standard first-order kinetic plot and given in Tables 3 and 4.

                  TABLE 3                                                         ______________________________________                                        Rate Constants for Uptake of Compound lb                                      by H9 Cells in 10% Fetal Calf Serum at 37° C.                          cpd. concentration (μM)                                                                     rate constant (s.sup.-1)                                     ______________________________________                                        1                3.6 ± 1.7 × 10.sup.-4                               5                3.3 ± 0.7 × 10.sup.-4                               25               2.5 ± 0.3 × 10.sup.-4                               ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Rate Constants for Uptake of Compound lb                                      by H9 Cells in 10% Fetal Calf Serum at 4° C.                           cpd. concentration (μM)                                                                     rate constant (s.sup.-1)                                     ______________________________________                                        1                3.5 ± 0.6 × 10.sup.-4                               5                2.7 ± 1.0 × 10.sup.-4                               25               2.3 ± 0.9 × 10.sup.-4                               ______________________________________                                    

Experiments on the effects of temperature and the presence of colchicineare summarized as: The total uptake of Compound 1b was decreased in thepresence of colchicine. The slope of the log/log plot of fluorescencevs. time shows essentially the same slope after 2 hours in the presenceand absence of colchicine; this supports the view that both passivediffusion across the lipid plasma membrane (rate unaffected bycolchicine) plus pinocytosis (poisoned by colchicine), contribute totakeup of Compound 1b. The protocol used is as above in (1), modified asbelow for those experiments in the presence of colchicine: Colchicine (1mg/ml) was added to H9 cells at 5×10.sup.≡ cells/ml, and incubated for 1hour. The cells were centrifuged, fresh medium was added, and 1b (25 μM)was added. Samples were taken at 0, 2, and 4 hours.

(2) Location Into Living Cells.

Following the protocol of (1), above, H9 cells were stained withCompound lb, and the technique of fluorescent microscopy was used todetermine visually the sites of localization of dye fluorescence inliving cells. Specifically, H9 cells were counted with a hemocytometerand diluted to 5×10⁵ /ml. Compound 1b was added to two aliquots to givedye concentrations of 5 and 25 μM, and the tubes were incubated for 1.5hours. Cytospin slides of the cell suspensions were made and they wereexamined with the fluorescence microscope (Olympus AH-2) using Bexcitation plus a 460 nm interference excitation filter and a 495 nmemission filter. The cells were also counted with a hemocytometer andtheir viability determined (see below). Both samples containing Compound1b were highly fluorescent with the cytoplasm being the primary site.The control slide showed no fluorescence at all. The cytoplasm displayeda veil-like pale green fluorescence plus bright yellow-green punctatesites of fluorescence. The former may reflect the diffusional componentof the dye incorporation, or may reflect binding in the ER membrane. Thelatter may indicate lysosomal localization of pinocytosed dye as occurswith Lucifer Yellow. The Compound 1b was rigorously excluded from thecell nucleus.

(3) Cell Viability in Absence of Light.

Cell viability was determined using dye incorporated according to theprotocol directly above. Viability was determined using a trypan bluedye exclusion test in which "live" cells with competent cell membranesexclude the dye. Table 5 summarizes the data obtained using cellsexposed to Compound 1b in culture medium, and Table 6 summarizes dataobtained using cells exposed to Compound VII.

                  TABLE 5                                                         ______________________________________                                        Viability of H9 Cells Following                                               Dark Exposure to Compound lb*                                                 (μM) cpd. lb                                                                          live cells dead cells                                                                              % viability                                   ______________________________________                                        0          136        2         98.6                                          5          116        4         96.7                                          25         136        4         97.1                                          ______________________________________                                         *Viability determined 2.5 hours after completion of exposure to dye in        medium for 1.5 hours.                                                    

                  TABLE 6                                                         ______________________________________                                        Viability of H9 Cells Following                                               Dark Exposure to Compound VII                                                 cpd. VII                                                                      (μM)  live cells   dead cells                                                                              % viability                                   ______________________________________                                        Viability determined 1 hour after completion of                               exposure to compound in 100% human plasma for 4 hours.                         1       280          12        95.9                                           5       286          16        94.7                                          25       339          16        95.5                                          Viability determined 1 hour after completion                                  of exposure to compound in medium for 24 hours                                 0       139          18        88.5                                           1       151          23        86.8                                          10       104          26        80.0                                          25       148          38        79.6                                          50       124          128       42.5                                          ______________________________________                                    

The results given in Tables 5 and 6 show that the dyes exhibit littledark toxicity in either the low or high protein and lipoproteinconcentrations of medium of whole human plasma, respectively.

(4) Dye-Mediated Light Kill of Living Cells.

Photoinduced killing of H9 cells with Compound 1b as the mediator ofphotochemical toxicity were carried out according to the followingprotocol:

Compound 1b was added to the H9 cells (5×10⁵ /ml) in medium or 100%human plasma to give dye concentrations of 0, 25 and 100 μM (1 control,2 duplicates of both non-azo dye samples). Two 2-ml aliquots of each ofthe duplicated dye samples was irradiated with 10J/cm² total energy of450±35 nm wavelength light in a sealed 3-ml test tube. Immediately afterirradiation, the cells were centrifuged at 450×g for 10 minutes, and thepellets resuspended in the original volume of fresh RPMI-1640+10%FCS+gentamycin. The cell suspensions were transferred to a 12-well TCplate and placed in a 37° C. 5% CO₂ incubator. Cell viability counts(trypan blue) were carried out at 4, 24, 48, and 72 hours postirradiation. The results of these experiments are summarized in Tables 7(medium) and 8 (100% human plasma).

                  TABLE 7                                                         ______________________________________                                        Photoinduced Kill of H9 Cells Mediated By                                     Compound lb in 10% FCS Medium                                                          Percent Viability                                                    Cpd. conc. (μM)                                                                       0        25           100                                          Time       no hν no hν hν no hν                                                                            hν                                ______________________________________                                         4         97.8     96.9     62.8  94.6  87.2                                 24         96.4     94.8      8.4  94.6  52.0                                 48         91.5     90.5     10.0  91.3  64.8                                 72         86.3     91.4     30.6  85.1  71.4                                 ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        Photoinduced Kill of H9 Cells Mediated by                                     Compound lb in 100% Human Plasma                                                       Percent Viability                                                    Cpd. conc. (μM)                                                                       0        25           100                                          Time       no hν no hν hν no hν                                                                            hν                                ______________________________________                                         4         100      97.2     97.9  99.2  97.4                                 24         97.0     93.4     94.4  94.3  92.4                                 48         91.9     86.4     86.5  90.7  92.6                                 72         81.3     85.0     90.2  87.4  88.2                                 ______________________________________                                    

Photoinduced killing of H9 cells with Compound VII as the mediator ofphotochemical toxicity were carried out in 10% FCS medium according tothe same general protocol using concentrations of the dye at 0, 5, 25and 100 μM., and irradiating at 420±5 nm at 10J/cm² total energy. Theresults of viability studies determined 4 hours after the procedure aregiven in Table 9.

                  TABLE 9                                                         ______________________________________                                        Photoinduced Kill of H9 Cells                                                 Mediated by Compound VII in 10% FCS Medium                                                Percent Viability                                                 Cpd. conc. (μM)                                                                          0      5          25   100*                                     ______________________________________                                        no hν      85.7   86.5       90.0 --                                       hν         88.2    0.0        0.0 1.8                                      ______________________________________                                         *Note:                                                                        The light from the filter was only able to reach approximately 2 mm into      the 100 μM irradiated sample due to absorption.                       

An additional study using DAUDI cells and lower concentrations ofCompound VII was performed according to the same protocol for studyingthe photochemical kill of H9 cells mediated by Compound VII. Viabilitywas determined by the trypan blue assay above, as well as by [³H]-thymidine incorporation. The [³ H]-thymidine incorporated protocolwas as follows:

After irradiation, the cells were diluted to 3×10⁵ cells/ml with mediumcontaining twice the normal concentration of gentamycin. The cells werepipetted into a 96-well TC plat (100 μl/well) and incubated at 37° C. ina 5% CO₂ incubator for 36 hours. At this time 1 μCi of [³ ]-thymidinewas added and allowed to incorporate for 6 hours. The cells were thenharvested on glass fiber filters and placed into scintillation vials towhich cocktail was added. The samples were counted on a Beckman model LS6000IC scintillation counter. The results are summarized in Table 10.

                  TABLE 10                                                        ______________________________________                                        Photoinduced Cell Kill of DAUDI Cells                                         Mediated by Compound VII in 10% FCS Medium                                                CPM                                                               Cpd. conc. (μM)                                                                          no hν    hν                                               ______________________________________                                        0             5.3 ± 0.2 × 10.sup.4                                                             2.5 ± 0.1 × 10.sup.4                       1             5.1 ± 0.3 × 10.sup.4                                                             1.2 ± 0.4 × 10.sup.2                       5             4.8 ± 0.3 × 10.sup.4                                                             1.1 ± 0.3 × 10.sup.2                       10            4.5 ± 0.2 × 10.sup.4                                                             1.0 ± 0.4 × 10.sup.2                       ______________________________________                                    

The results tabulated in Tables 8, 9 and 10 show that Compound VII is apotent mediator of photochemical toxicity, and a highly efficientphotochemical cell inactivator at concentrations as low as 1 μM. usinglight energy fluxes in the range of 10J/cm².

(5) Encapsulation of Hemoglobin.

Bovine hemoglobin was purified by the addition of 3 volumes of deionizedwater to bovine blood obtained from a local slaughter house followed bygentle agitation for one hour. Cell debris was removed by centrifugationat 18,000 xg for 15 minutes. No further purification of the hemoglobinwas attempted for these studies. This hemoglobin solution was warmed to40C on a stirring hotplate and 0.10 volume of lipid/dye solution slowlyinjected into the hemoglobin solution through a septum on an invertedflask. The lipid/dye solution consisted of 10 mg lecithin, 2 mggramicidin, and 1 ml of 1.1 mg/ml dye dissolved in Cremophor RH 40 alldissolved in ethanol. Sonication was used to suspend all lipid. Afterthe solution had cooled to room temperature, the vesicles were dialyzedovernight against 0.325M Tris-Cl⁻ pH 8.0. Free hemoglobin was removedfrom the liposomes by gel filtration over G-50 and subsequent passageover an A-25 anion exchange column equilibrated with 32 mM Tris-Cl⁻ pH8.5. All manipulations were performed under fluorescent lighting.Further exposure to light from a Xenon arc lamp did not increase thestability of the preparation. Vesicle samples were lyophilized andsubsequently resuspended in deionized water.

(6) Effect of Compound VII on Viruses.

Human Herpes Simplex Virus Type I (HSV-I). Cell-free and serum-freeHSV-I were suspended in Liebowitz medium at nominal PFU values of 5×10⁵/ml. Compound VII was dissolved in chremophor to give a 20 μM stocksolution, and aliquots of the dye stock solution were dissolved in viralsuspensions to give dye concentrations varying between 78 nM and 10 μM.One-ml aliquots of viral suspension containing Compound VII were placedin 35 mm diameter Petri dishes and irradiated with 420±5 nm lightfiltered from a xenon arc lamp for 9 min 10 sec to give 20J/cm² lightenergy flux. Standard plaque assay for vial infectivity gave survivaldata tabulated in Table 11, below (% survival=[infectivity of irradiatedvirus/infectivity of dark control]).

                  TABLE 11                                                        ______________________________________                                        Photoinduced Viral Inactivation Mediated by Compound 1a                       Infectivity                                                                                                            log.sub.10                           Cpd.                              %      reduc-                               conc. (μM)                                                                         PFU/ml   PFU/ml   Average Survival                                                                             tion                                 ______________________________________                                        0 (control)                                                                           5.5 × 10.sup.5                                                                   7.0 × 10.sup.5                                                                   6.25 × 10.sup.5                               10      0        0        0       <0.0002                                                                              >5.7                                 5       0        0        0       <0.0002                                                                              >5.7                                 2.5     0        0        0       <0.0002                                                                              >5.7                                 1.25    0        0        0       <0.0002                                                                              >5.7                                 0.625   0        --       0       <0.0002                                                                              >5.7                                 0.312   10       5        7.5      0.0012                                                                               4.9                                 0.156   20       15       17.5     0.0028                                                                               4.5                                 0.078   45       30       37.5     0.006  4.2                                 ______________________________________                                    

Bovine Herpes Virus Type I (BHV-1). Cell-free and serum-free virus(BHV-1 reference strain from Dr. M. L. Vickers, ADL SDSU) was purifiedby potassium tartrate gradient ultracentrifugation. Virus was thenresuspended in minimal buffer. Compound VII was diluted with ethanol toa concentration of 5 mg/ml. The dye stock solution was prepared bysequentially adding the ethanolic dye solution to fetal bovine serum(FBS), and diluting the dye-FBS mixture with MEM to give 10% FBS in MEM,so that the final concentration of the compound was 500 ng/ml. Alldilutions of the dye in the medium were made from this stock solution.Dye/virus mixtures were all prepared as 1:1 mixtures of virus and dyestock solution. The virus was incubated for 30 min at 37° C. prior toirradiation, and then irradiated using a xenon arc lamp to give 10J/cm²total irradiative flux. Photomodified virus was then incubated with theappropriate cells (fetal bovine lung - FBL - or MDBK) in 10% FBS in MEMfor 1 hour. Then unbound virus was taken off, the cells were washed withCa/Mg-free PBS, and the cells were then placed in 10% FBS-MEM. The cellswere incubated for 24 hours at 37° C. in a 5% CO₂ incubator. After 24and 48 hours, the cells were assayed for cytophathic effect (CPE), andthe results expressed in terms of using TCID₅₀ units. The viralinactivation data are expressed in terms of % inactivation in Table 12.

                  TABLE 12                                                        ______________________________________                                        Photoinactivation of Bovine Herpes Virus Type I                               Mediated by Compound VII                                                      % Inactivation                                                                Cpd. conc. (μM)                                                                       24 h      log.sub.10 red.                                                                        48 h    log.sub.10 red.                         ______________________________________                                        0          0                  0                                               0.035      99.4      3.2      89.9    0.95                                    0.070      99.8      3.7      98.7    2.0                                     0.14       >99.98    >3.7     99.4    3.2                                     0.27       >99.98    >3.7     99.4    3.2                                     0.54       >99.98    >3.7     >99.98  >3.7                                    1.09       >99.98    >3.7     >99.98  >3.7                                    ______________________________________                                    

Surface Antigen Assays The BHV-1 virus prepared as above (20480 TCID₅₀)was (i) held as control; (ii) treated with dye (concentrations from 7.5to 250 ng/ml) in cell culture medium; or (iii) treated with Compound VIIand light-treated in cell culture medium. All virus was plated in96-well flat bottom plates for 24 hours in 5x replicates on FBL cells.At that time the wells were examined for CPE, scored, and 2x wells werefixed in acetone and paraformaldehyde for examination of intracellularand surface BHV-1 antigen, respectively. The cells were treated with 50μl of an anti-BHV-1 monoclonal antibody (gift of Dr. J. Collins,Diagnostic Laboratory, Colorado State University, School of VeterinaryMedicine) used as a common diagnostic reagent, for 30 minutes at roomtemperature. The cells were further stained with either FITC-labeledanti-mouse or biotinylated anti-mouse and avidin rPE. The samples wereplaced on slides and read by fluorescent microscopy using a 495 nmexcitor filter and 525 long pass emission filter on a Leitzepifluorescent microscope. The results were as follows:

Control virus--Both surface and internalized viral antigen was observedin the infected cells and most cells had clearly demonstrable internalantigen.

Virus and dye (dark control)--Much surface viral antigen was detected atall dye concentrations. An interesting observation was that when the rPEwas used, an intermediate color to the probe red and the yellow-green ofCompound VII was observed, as if FET was occurring. Internal antigen wasobserved and correlated with CPE levels. Cells fixed with acetoneallowed for examination of the viral antigen, but the fixation removedthe Compound VII.

photochemically attenuated virus--The results were similar to the darkcontrol. Surface antigen was observed at all concentrations of CompoundVII, and internal antigen was observed at all concentrations of CompoundVII which showed CPE. The same color effect was seen in paraformaldehydefixed cells.

(7) Effect Of Ed6Br (Compound VII) And Light On Aqueous Suspensions OfHerpes Simplex Virus Type 1

Herpes simplex virus type 1 (HSV-1), the MacIntyre strain, was purchasedfrom the American Type Culture Collection (ATCC) and propagated in Verocells (ATCC) to a concentration of 10⁶ -10⁷ plaque forming units (PFU)per milliliter (ml). This solution was used as stock virus. A volume of0.1-0.5 ml of stock virus was added to separate aliquots of modifiedLeibowitz medium (L₁₅ medium, Whittaker MA Bioproducts, and containing2.5% or less of serum proteins) in order to give a PFU concentrationbetween 10⁵ and 10⁷.

Ed6Br in Cremaphor EL (approximately 2.5 millimolar) was added toduplicate tubes of the virus-medium mixture. The concentrations of Ed6Bremployed were 0.78, 0.156, 0.312, 0.625, 1.25, 2.5, 5.0 and 10.0micromolar. Duplicate tubes representing each concentration of Ed6Br inthe medium-virus mixture were irradiated by light at a wave length ofapproximately 420 nm with a fluence of about 20 J/cm². Approximately30-60 min. elapsed between the addition of each concentration of Ed6Brand exposure to light. During the holding period, the samples weremaintained at 4° C. Except during the time of irradiation, allmanipulations were carried out with minimal exposure to extraneouslight. A sample of virus-medium mixture but not containing Ed6Br wasalso irradiated under the same conditions. During the holding period andthe period of irradiation, the duplicate samples of virus and mediumcontaining different concentrations of Ed6Br were held in the dark. Inaddition, samples of the three different samples containing differentconcentrations of Ed6Br and a sample of the virus-medium mixture withoutEd6Br were also maintained in the dark.

Each sample including the stock virus were assayed on Vero cells forPFU/ml of HSV-1. The assay consisted of growing Vero cells in minimalessential medium with Hanks balanced salt solution supplemented with 10%fetal bovine serum, L-glutamine and antibiotics. Hepes buffer (2%) wasadded for growth in open plates (twelve well microplates from CO-star).Ten fold dilutions of each sample were prepared and 0.1 ml of theappropriate dilution for each individual sample was adsorbed at 37° C.for 1.5 hr. on a cell monolayer from which the growth medium had beenremoved. After the adsorption period, the cell monolayer was washed andan overlay consisting of equal volumes of 2X strength L-15 medium and 2%methylcellulose was added. Following an appropriate incubation time at37° C. (about 4 days), the overlay medium was removed. Monolayers werefixed with methanol and stained with giemsa to elaborate the presence ofplaques. The plaques were counted using a dissecting microscope at amagnification of 20X.

The results of the experiment can be observed in Table 13. It is clearthat Ed6Br in a concentration as low as 0.078 mM, in combination withlight at a wavelength of 420 # 5 nm having an energy density of 20J/cm2, achieved a near total (larger than 99.99%) kill of HSV-1 inaqueous medium containing 2.5% or less of serum proteins. The use oflarger amounts of ED6Br resulted in a similar reduction in viralinfectivity. Viral-medium suspension samples containing the 8 differentconcentrations but which were not irradiated showed no evidence ofcellular toxicity when assayed in the Vero cells as described above.

                  TABLE 13                                                        ______________________________________                                        Inactivation of HSV-1 with Ed6Br (in Cremophor)                               and 20 J/cm2 of Light at 420 nm                                                                                  Log.sub.10                                 Sample        Irradiation                                                                              PFU/ml    Reduction                                  ______________________________________                                        HSV Control   Yes        6.3 × 10.sup.5                                 HSV + 10 μM Ed6Br                                                                        Yes        0         5.799                                      HSV + 5 μM Ed6Br                                                                         Yes        0         5.799                                      HSV + 2.5 μM Ed6Br                                                                       Yes        0         5.799                                      HSV + 1.25 μM Ed6Br                                                                      Yes        0         5.799                                      HSV + .625 μM Ed6Br                                                                      Yes        0         5.799                                      HSV + .312 μM Ed6Br                                                                      Yes        7.5 × 10.sup.0                                                                    4.924                                      HSV + .156 μM Ed6Br                                                                      Yes        1.8 × 10.sup.1                                                                    4.544                                      HSV + .078 μM Ed6Br                                                                      Yes        3.8 × 10.sup.1                                                                    4.219                                      ______________________________________                                         PFU: Plaque forming unit.                                                     HSV-1: Diluted 1:10 in medium yielded 6.0 × 10.sup.5 PFU/ml.            Virus diluted in blood and not irradiated yielded 6.3 × 10.sup.5        PFU/ml.                                                                  

(8) Protein Cross-Linking With DiEd6Br (Compound XLIV) And Light

Gel electrophoresis studies of various individual protein preparationscontaining DiEd6Br have demonstrated the formation of slowerelectromigrating, thus heavier, molecular species upon photoirradiationof solutions containing both protein and the dye with light of nominally420 nm wavelength and the complete absence of these species in likeprotein sample controls with or without DiEd6Br in the absence of light.Additional control with photoillumination showed absence of the heavierspecies in the absence of DiEd6Br. Concomitant with the appearance ofthe heavier species with photoillumination with the decrease inintensity of electromigrated bands of the initially present protein.Additionally, comparison of molecular weights of the newly appearingspecies deduced from electromigration distance showed the new bands tohave molecular weights closely approximating integral multiple values(e.g., 2X, 3X, 4X, . . . ) of the value for the parent basic structuralprotein unit; e.g., monomer. This approximately integral multiplemolecular weight relationship of the initial protein monomers and theheavier species formed upon photoillumination strongly suggested thecrosslinking of two or more protein monomers by DiEd6Br upon reaction ofeach terminal naphthalamide moiety in the metastable states withnucleophilic residues of the pairwise linked proteins.

Results of experiments demonstrating crosslinking of proteins in thepresence of DiEd6Br upon photoillumination with filtered light of 420 #5 nm wavelength are summarized in Table 14. In these experiments, thelipophilic DiEd6Br was introduced into the aqueous suspensions of theproteins through the use of Cremophor EL micelles. During equilibrationprior to light exposure, the photochemical transferred to thehydrophobic environments of the viral envelope of vesicular stomatitisvirus (VSV) and lipid intenor of high density lipoprotein (HDL) viacollisional contact with the dye bearing micelles and via similarcollision to the more hydrophobic surface regions of the proteins ofF-actin polymers and albumin dissolved in aqueous solution.

                  TABLE 14                                                        ______________________________________                                        Protein Crosslinking with DiED6Br                                             ______________________________________                                        Part A: Results                                                                           Protein           Inferred                                                    Monono-  Gel      Crosslinked                                                                           Gel                                     Systems     mers     mw (kD)  Species mw (kD)                                 ______________________________________                                        1.  Hydrophobic-lipid membrane with DiEd6Br delivered in                          Cremophor EL (ethoxylated caster bean oil) micellar                           suspensions.                                                              (a)   Human     Apopro-  17.8   8 Apopro-                                                                             141                                         High      tein I          tein I units                                        Density                                                                       Lipoprotein                                                                   (HDL)                                                                   (b)   Vesicular G-pro-   63 (incl.                                                                            2 G-protein                                                                           130                                         stomatitis                                                                              tein (en-                                                                              carbo- units or                                            Virus     velope   hydrate                                                                              1 G-pro-                                            (Ogden)   glycopro-                                                                              moeity)                                                                              tein + 2                                            strain)   tein)           M-protein                                           (VSV)                     units                                         Plus many ill separated heavier bands merging together near                   origin.                                                                       2.  Hydrophobic regions within proteins with DiEd6Br                              delivered in Cremophor EL (ethoxylated castor bean oil)                       micellar suspension.                                                      (a)   F-actin   G-actin  47     2 G-actin                                                                              94                                         polymers of                                                                             mono-           monomers                                            G-actin in                                                                              mers            3 G-actin                                                                             141                                         100 nM                    monomers                                            KCl                                                                     Plus many ill separated heavier bands merging together near                   origin.                                                                        (b)  Human     HSA      64     2 HSA   120                                         serum     monomer         units                                               Albumen                   3 HSA   180                                         (HSA)                     units                                               in                        3 HSA   240                                         Phosphate                 units                                               buffered                                                                      saline                                                                        pH = 7.2                                                                Plus many ill separated heavier bands merging together near                   origin.                                                                       ______________________________________                                        Part B: Methods Summary                                                                 Concentration         Gel                                           System      Protein  DiEd6Br  Fluence.sup.(3)                                                                       System.sup.(4)                          ______________________________________                                        1.  Hydrophobic-lipid membrane                                                (a)   Human     10 mg/   25.0 μM                                                                           400 J/cm.sup.2                                                                        SDS-                                        High      ml.sup.(1)              PAGE                                        Density                                                                       Lipopro-                                                                      tein (HDL)                                                              (b)   Vesicular 300-400  10.0 μM                                                                           460 J/cm.sup.2                                                                        SDS-                                        Stomatitis                                                                              μg/ml                PAGE                                        Virus     (approx-                                                            (VSV)     imately                                                                       1.5 μM                                                                     in both G                                                                     and M                                                                         proteins)                                                     2.  Hydrophobic Protein Regions                                               (a)   F-actin   85.7     40.0 μM                                                                           400 J/cm.sup.2                                                                        SDS-                                                  μM.sup.(2)           PAGE                                  (b)   Human     60.0 μM                                                                             30.0 μM                                                                           791 J/cm.sup.2                                                                        SDS-                                        Serum                             PAGE                                        Albumen                                                                 ______________________________________                                         .sup.(1) Total HDL concentration.                                             .sup.(2) G-actin monomer concentration.                                       .sup.(3) 420 ± 5 nm wavelengths filtered from 2 xeonon arc lamps at an     irradiance of 20 mW/cm2.                                                      .sup.(4) Proteins were dissolved in sodium dodecyl sulfate (SDS) and          electrophoresed on polyacrylamide gradient gels (7-11%) and stained with      silver.                                                                  

(9) Effect Of DiEd6Br (Compound XLIV) And Light On Aqueous SuspensionsOf Herpes Simplex Virus Type 1

Herpes simplex virus type 1 (HSV-1), the MacIntyre strain, was purchasedfrom the American Type Culture Collection (ATCC) and propagated in Verocells (ATCC) to a concentration of 10⁶ -10⁷ plaque forming units (PFU)per milliliter (ml). This solution was used as stock virus. A volume of0.1-0.5 ml of stock virus was added to separate aliquots of modifiedLeibowitz medium (L₁₅ medium, Whittaker MA Bioproducts, and containing2.5% or less of serum proteins) in order to give a PFU concentrationbetween 10⁵ and 10⁷.

DiEd6Br in Cremophor EL (approximately 2.5 millimolar) was added toduplicate tubes of the virus-medium mixture. The concentrations ofDiEd6Br employed were 0.0099, 0.0195, 0.039, 0.78, 0.156, 0.312, 0.625,1.25, 2.5, 5.0 and 10.0 micromolar. Duplicate tubes representing eachconcentration of DiEd6Br in the medium-virus mixture were irradiated bylight at a wave length of approximately 420 nm with a fluence of about20 J/cm². Approximately 30-60 min elapsed between the addition of eachconcentration of DiEd6Br and exposure to light. During the holdingperiod, the samples were maintained at 4° C. Except during the time ofirradiation, all manipulations were carried out with minimal exposure toextraneous light. A sample of virus-medium mixture but not containingDiEd6Br was also irradiated under the same conditions. During theholding period and the period of irradiation, the duplicate samples ofvirus and medium containing different concentrations of DiEd6Br wereheld in the dark. In addition, samples of the three different samplescontaining different concentrations of DiEd6Br and a sample of thevirus-medium mixture without DiEd6Br were also maintained in the dark.

Each sample including the stock virus were assayed on Vero cells forPFU/ml of HSV-1. The assay consisted of growing Vero cells in minimalessential medium with Hanks balanced salt solution supplemented with 10%fetal bovine serum, L-glutamine and antibiotics. Hepes buffer (2%) wasadded for growth in open plates (twelve well microplates from Co-star).Ten fold dilutions of each sample were prepared and 0.1 ml of theappropriate dilution for each individual sample was adsorbed at 37° C.for 1.5 hr. on a cell monolayer from which the growth medium had beenremoved. After the adsorption period, the cell monolayer was washed andan overlay consisting of equal volumes of 2X strength L-15 medium and 2%methylcellulose was added. Following an appropriate incubation time at37° C. (about 4 days), the overlay medium was removed. Monolayers werefixed with methanol and stained with giemsa to elaborate the presence ofplaques. The plaques were counted using a dissecting microscope at amagnification of 20X.

The results of the experiment can be observed in Table 15. It is clearthat DiEd6Br in a concentration as low as 0.0195 mM, in combination withlight at a wavelength of 420 # 5 nm having an energy density of J/cm²,achieved a near total (larger than 99.99%) kill of HSV-1 in aqueousmedium containing 2.5% or less of serum proteins. The use of largeramounts of DiEd6Br resulted in a similar reduction in viral infectivity.Viral-medium suspension samples containing the 8 differentconcentrations of DiEd6Br but which were not irradiated showed noevidence of cellular toxicity when assayed in the Vero cells asdescribed above.

                  TABLE 15                                                        ______________________________________                                        Inactivation of HSV-1 with DiEd6BR (in Cremophor EL)                          and 20 J/cm2 of Light at 420 nm                                                                                  Log.sub.10                                 Sample          Irradiation                                                                             PFU/ml   Reduction                                  ______________________________________                                        HSV Control     No        1.3 × 10.sup.6                                HSV + 10 μM DiEd6Br                                                                        No        1.2 × 10.sup.6                                                                   0.0348                                     HSV + 2.5 μM DiEd6Br                                                                       No        1.3 × 10.sup.6                                                                   0.0                                        HSV + .625 μM DiEd6Br                                                                      No        1.0 × 10.sup.6                                                                   0.1139                                     HSV + .156 μM DiEd6Br                                                                      No        1.1 × 10.sup.6                                                                   0.0725                                     Test #1                                                                       HSV Control     Yes       9.9 × 10.sup.5                                HSV + 10 μM DiEd6Br                                                                        Yes       0        5.9956                                     HSV + 2.5 μM DiEd6Br                                                                       Yes       0        5.9956                                     HSV + .625 μM DiEd6Br                                                                      Yes       0        5.9956                                     HSV + .156 μM DiEd6Br                                                                      Yes       0        5.9956                                     Test #2                                                                       HSV Control     Yes       7.4 × 10.sup.5                                HSV + .156 μM DiEd6Br                                                                      Yes       0        5.8692                                     HSV + .078 μM DiEd6Br                                                                      Yes       0        5.8692                                     HSV + .039 μM DiEd6Br                                                                      Yes       9.9 ×  10.sup.0                                                                  4.8736                                     HSV + .0195 μM DiEd6Br                                                                     Yes       4.3 × 10.sup.1                                                                   4.2358                                     HSV + .0099 μM DiEd6Br                                                                     Yes       4.0 × 10.sup.2                                                                   3.2672                                     ______________________________________                                         PFU: Plaque forming unit.                                                     HSV-1: Diluted 1:10 in medium yielded 1.3 × 10.sup.5 PFU/ml.            Virus diluted in blood and not irradiated yielded 1.0 × 10.sup.5        PFU/ml.                                                                  

(10) Effect Of DiEd6Br (Compound XLIV) And Light On Aqueous SuspensionsOf Herpes Simplex Virus Type 1 Containing 15% Fetal Calf Serum

Herpes simplex virus type 1 (HSV-1), the MacIntyre strain, was purchasedfrom the American Type Culture Collection (ATCC) and propagated in Verocells (ATCC) to a concentration of 10⁶ -10⁷ plaque forming units (PFU)per milliliter (ml). This solution was used as stock virus. A volume of0.1-0.5 ml of stock virus was added to separate aliquots of modifiedLeibowitz medium (L15 medium, Whittaker MA Bioproducts) containingapproximately 15% fetal calf serum in order to give a PFU concentrationbetween 10⁵ and 10⁷.

DiEd6Br in Cremaphor EL (Ethoxylated castor-bean oil, approximately 2.5millimolar) was added to duplicate tubes of the virus-medium mixture.The concentrations of DiEd6Br employed were 0.078, 0.156, 0.312, 0.625,1.25, 2.5, 5.0 and 7.5 micromolar. Duplicate tubes representing eachconcentration of DiEd6Br in the medium-virus mixture were irradiated bylight at a wave length of approximately 420 nm with a fluence of about

20 J/cm². Approximately 30-60 minutes elapsed between the addition ofeach concentration of DiEd6Br and exposure to light. During the holdingperiod, the samples were maintained at 4° C. Except during the time ofirradiation, all manipulations were carried out with minimal exposure toextraneous light. A sample of virus-medium mixture but not containingDiEd6Br was also irradiated under the same conditions. During theholding period and the period of irradiation, the duplicate samples ofvirus and medium containing different concentrations of DiEd6Br wereheld in the dark. In addition, samples of the three different samplescontaining different concentrations of DiEd6Br and a sample of thevirus-medium mixture without DiEd6Br were also maintained in the dark.

Each sample including the stock virus were assayed on Vero cells forPFU/ml of HSV-1. The assay consisted of growing Vero cells in minimalessential medium with Hanks balanced salt solution supplemented with 10%fetal bovine serum, L-glutamine and antibiotics. Hepes buffer (2%) wasadded for growth in open plates (twelve well microplates from CO-star).Ten fold dilutions of each sample were prepared and 0.1 ml of theappropriate dilution for each individual sample was adsorbed at 37° C.for 1.5 hr. on a cell monolayer from which the growth medium had beenremoved. After the adsorption period, the cell monolayer was washed andan overlay consisting of equal volumes of 2X strength L-15 medium and 2%methylcellulose was added. Following an appropriate incubation time at37° C. (about 4 days), the overlay medium was removed. Monolayers werefixed with methanol and stained with giemsa to elaborate the presence ofplaques. The plaques were counted using a dissecting microscope at amagnification of 20X.

The results of the experiment can be observed in Table 16. It is clearthat DiEd6Br in a concentration as low as 0.156 mM, in combination withlight at a wavelength of 420 # 5 nm having an energy density of 20J/cm², achieved a near total (larger than 99.99%) kill of HSV-1 inaqueous medium containing 2.5% or less of serum proteins. The use oflarger amounts of DiEd6Br resulted in a similar reduction in viralinfectivity. Viral-medium suspension samples containing the 8 differentdye concentrations but which were not irradiated showed no evidence ofcellular toxicity when assayed in the Vero cells as described above.

                  TABLE 16                                                        ______________________________________                                        Inactivation of HSV-1 in 15% Fetal Calf Serum with                            DiEd6Br (in Cremophor) and 20 J/cm2 of Light at 420 nm                                                           Log.sub.10                                 Sample         Irradiation                                                                             PFU/ml    Reduction                                  ______________________________________                                        HSV Control    Yes       1.0 × 10.sup.5                                 HSV + 10 μM DiEd6Br                                                                       Yes       0         ≧5                                  HSV + 5 μM DiEd6Br                                                                        Yes       0         ≧5                                  HSV + 2.5 μM DiEd6Br                                                                      Yes       0         ≧5                                  HSV + 1.25 μM DiEd6Br                                                                     Yes       0         ≧5                                  HSV + .625 μM DiEd6Br                                                                     Yes       6.0 × 10.sup.1                                                                    4.0                                        HSV + .312 μM DiEd6Br                                                                     Yes       8.5 × 10.sup.1                                                                    3.1                                        HSV + .156 μM DiEd6Br                                                                     Yes       4.5 × 10.sup.3                                                                    1.35                                       HSV + .078 μM DiEd6Br                                                                     Yes       4.2 × 10.sup.4                                                                    0.38                                       ______________________________________                                         PFU: Plaque forming unit.                                                     HSV-1: Diluted 1:10 in medium yielded 1.0 × 10.sup.5 PFU/ml.       

(11) Effect Of Prior Activation Of Ed6Br (Compound VII) With 420 nmLight On Herpes Simplex Virus Type 1

Herpes simplex virus type 1 (HSV-1), the MacIntyre strain, was purchasedfrom the American Type Culture Collection (ATCC) and propagated in Verocells (ATCC) to a concentration of 2.7×10⁵ plaque forming units (PFU)per milliliter (ml). This solution was used as stock virus.

Ed6Br in Cremophor EL was added to four separate aliquots of modifiedLiebowitz medium (L-15 medium, Whittaker MA Bioproducts) to giveconcentrations of 25 mM (2 aliquots) and 100 mM (2 aliquots),respectively, of the 1,8-naphthalimide. Two of the aliquots with 25 and100 micromolar dye concentration, respectively, were irradiated with 420# 5 nm filtered radiation from a Xenon arc lamp at an irradiance of 20mW/cm² for 166 minutes to give a total light fluence of 200 J/cm². Theremaining two aliquots at 25 and 100 mM concentration were handled induplicate fashion with aluminum foil wrapping but not illuminated.

Immediately after illumination 100 microliter of stock HSV-1 suspensionat 2.3×10⁵ PFU/ml was added to each of the four samples containing the1,8-naphthalimide compound. Each sample was held in the dark at 4° C.for 24 hr. prior to infectivity assay.

Each sample including the stock virus were assayed on Vero cells forPFU/ml of HSV-1. The assay consisted of growing Vero cells in minimalessential medium with Hanks balanced salt solution supplemented with 10%fetal bovine serum, L-glutamine and antibiotics. Hepes buffer (2%) wasadded for growth in open plates (twelve well microplates from CO-star).Ten fold dilutions of each sample were prepared and 0.1 ml of theappropriate dilution for each individual sample was adsorbed at 37° C.for 1.5 hr. on a cell monolayer from which the growth medium had beenremoved. After the adsorption period, the cell monolayer was washed andan overlay consisting of equal volumes of 2X strength L-15 medium and 2%methylcellulose was added. Following an appropriate incubation time at37° C. (about 4 days), the overlay medium was removed. Monolayers werefixed with methanol and stained with giemsa to elaborate the presence ofplaques. The plaques were counted using a dissecting microscope at amagnification of 20X.

The results of the experiment can be observed in Table 17. It is clearthat Ed6Br in a concentration as low as 100 mM, with activation usinglight at a wavelength of 420 # 5 nm having an energy density of 20J/cm², prior to addition to the viral suspension, achieved a near total(larger than 99.99%) kill of HSV-1 in aqueous medium containing 2.5% orless of serum proteins. The viral-medium suspension samples containingthe two different concentrations but which were not previouslyirradiated showed significantly less cellular toxicity than theirradiated samples when assayed in the Vero cells as described above.

                  TABLE 17                                                        ______________________________________                                        Inactivation of HSV-1 with Previously Activated Ed6Br                                       Prior               Log.sub.10                                  Sample        Activation PFU/ml   Reduction                                   ______________________________________                                        HSV stock     -          2.7 × 10.sup.5                                                                   --                                          HSV + 25 μM Ed6Br                                                                        +          8.4 × 10.sup.3                                                                   1.51                                        HSV + 25 μM Ed6Br                                                                        -          4.5 × 10.sup.4                                                                   0.78                                        HSV + 100 μM Ed6Br                                                                       +          3.0 × 10.sup.1                                                                   3.95                                        HSV + 100 μM Ed6Br                                                                       -          9.0 × 10.sup.3                                                                   1.48                                        ______________________________________                                    

(12) Effect Of DiEd6Br (Compound XLIV) And Light On Aqueous SuspensionsOf Herpes Simplex Virus Type 1 Containing 15% Fetal Calf Serum And HumanBlood Platelets

Herpes simplex virus type 1 (HSV-1), the MacIntyre strain, was purchasedfrom the American Type Culture Collection (ATCC) and propagated in Verocells (ATCC) to a concentration of 10⁶ -10⁷ plaque forming units (PFU)per milliliter (ml). This solution was used as stock virus. A volume of0.1-0.5 ml of stock virus was added to separate aliquots of modifiedLeibowitz medium (L15 medium, Whittaker MA Bioproducts) containing 15%added fetal calf serum proteins and human blood platelets at 2×10⁹ perml of viral suspension in order to give a PFU concentration between 10⁵and 10⁷.

DiEd6Br in Cremaphor EL (approximately 2.5 millimolar) was added toduplicate tubes of the virus-medium mixture. The concentrations ofDiEd6Br employed were 0.156 and 0.625 micromolar. Duplicate tubesrepresenting each concentration of DiEd6Br in viral suspension wereirradiated by light at a wave length of approximately 420 nm withfluence values of about 5, 10, 20, 40, and 60 J/cm². Approximately 30-60min. elapsed between the addition of each concentration of DiEd6Br andexposure to light. During the holding period, the samples weremaintained at 4° C. Except during the time of irradiation, allmanipulations were carried out with minimal exposure to extraneouslight. A sample of virus-medium mixture but not containing DiEd6Br wasalso irradiated under the same conditions. During the holding period andthe period of irradiation, the duplicate samples of virus and mediumcontaining different concentrations of DiEd6Br were held in the dark. Inaddition, samples of the three different samples containing differentconcentrations of DiEd6Br and a sample of the virus-medium mixturewithout DiEd6Br were also maintained in the dark.

Each sample including the stock virus were assayed on Vero cells forPFU/ml of HSV-1. The assay consisted of growing Vero cells in minimalessential medium with Hanks balanced salt solution supplemented with 10%fetal bovine serum, L-glutamine and antibiotics. Hepes buffer (2%) wasadded for growth in open plates (twelve well microplates from CO-star).Ten fold dilutions of each sample were prepared and 0.1 ml of theappropriate dilution for each individual sample was adsorbed at 37° C.for 1.5 hr. on a cell monolayer from which the growth medium had beenremoved. After the adsorption period, the cell monolayer was washed andan overlay consisting of equal volumes of 2X strength L-15 medium and 2%methylcellulose was added. Following an appropriate incubation time at37° C. (about 4 days), the overlay medium was removed. Monolayers werefixed with methanol and stained with giemsa to elaborate the presence ofplaques. The plaques were counted using a dissecting microscope at amagnification of 20X.

The results of the experiment can be observed in Table 18. It is clearthat DiEd6Br in a concentration as low as 0.625 mM, in combination withlight at a wavelength of 420 # 5 nm having an energy density of 60J/cm², achieved a near total (larger than 99.86%) kill of HSV-1 inaqueous medium containing 15% of serum proteins and platelets at typicalconcentrations during blood bank storage. Viral suspension samplecontaining the 2 different concentrations and DiEd6Br but which were notirradiated showed no evidence of cellular toxicity when assayed in theVero cells as described above.

                  TABLE 18                                                        ______________________________________                                        Inactivation of HSV-1 with DiEd6Br (in                                        Cremophor EL) and 420 nm Light in 15% Fetal                                   Calf Serum Containing Human Platelets                                                    Light Fluence           Log.sub.10                                 Sample     (J/cm.sup.2)                                                                              PFU/ml      Reduction                                  ______________________________________                                        HSV Control                                                                              No          7.6 × 10.sup.7                                                                      --                                         HSV + 625 μM                                                                           5          5.0 × 10.sup.5                                                                      2.18                                       DiEd6Br    10          4.5 × 10.sup.5                                                                      2.22                                                  20          2.0 × 10.sup.4                                                                      3.58                                                  40          1.1 × 10.sup.3                                                                      4.84                                                  60          None detected                                                                             7                                          HSV + 0.156 μM                                                                         5          7.4 × 10.sup.7                                                                      0.01                                       DiEd6Br    10          6.0 × 10.sup.7                                                                      0.10                                                  20          1.5 × 10.sup.7                                                                      0.70                                                  40          4.5 × 10.sup.6                                                                      1.22                                                  60          1.5 × 10.sup.6                                                                      1.70                                       ______________________________________                                         PFU: Plaque Forming Unit                                                 

(13) Effect Of DiEd6Br (Compound XLIV) And Light On Aqueous SuspensionsOf Human Immunodeficiency Virus

Human immunodeficiency virus (HIV) was propagated in the CEM Tlymphoblastoid line A:301 as described previously by Chanh et al (1986).Aliquots of cell-free HIV were prepared by centrifugation andresuspension in RPMI cell culture medium with a concentration of 1×10⁵IU (infectious units) per ml. Two aliquots having no DiE6Br but onebeing exposed to light served as E6Br free controls. DiE6Br was added to6 additional aliquots of viral suspension to give concentrations of1.25, 2.50, and 5.0 mM in each of a pair of samples, one member of eachpair serving as the light free control. Following 30 min. delay, onesample at each of the listed DiE6Br concentrations was exposed at anirradiance of 20 mW/cm² to give a light fluence of 20 J/cm² of 420 # 5nm radiation filtered from a xenon arc lamp, the other sample serving asdark control. After illumination 50 mL from each of the samples wastransferred to individual wells of a 96-well flat bottomed plate intriplicate. To each sample well 50 mL of a suspension containing 5.0×10⁵MT-4 cells per ml are transferred and the culture plates incubated at37.5° C. in a 50% CO₂ atmosphere. On the fourth day of incubation 100 mLof culture supernatant are removed and replaced with 100 mL of RPMI-1640with 10% fetal calf serum.

Upon the seventh day of culture the infectivity of HIV is assayed usingthe MTT stain procedure which monitors the viability of the MT-4 cells.Viability of cell cultured with a treated viral sample is compared tothat of an untreated control triplicate. Ten mL of the viability stainMTT [3-(4,5-dimethyl thiazole-2-yl) -2,5-diphenyltetrazolium bromide] at5 mg/ml concentration in phosphate buffered saline (pH 7.3) is addeddirectly to the MT-4 cells in culture. Cells are incubated for 4 hrs.after addition of the stain and 1.25 mL of 0.094N HCl in nopropanol isadded to develop the stain. The optical density of the layer of stainedMT-4 cells lining the bottom of the sample well is read at 570 nm with astandard microplate reader (Coulter Company) and reading three emptywells as air blanks.

The results of the experiment can be viewed in Table 19. The essentiallycomplete inactivation of HIV at DiEd6Br concentrations of 1.25 mM orgreater is evident in the clearly approximate equality of the measuredoptical density read in virus free controls and the light treatedsamples containing DiEd6Br.

                  TABLE 19                                                        ______________________________________                                        Inactivation of Aqueous HIV Suspension                                        with DiEd6Br and 420 nm Light                                                        DiEd6Br                                                                Sample Concentration Light Fluence                                                                             Optical                                      Number (μM)       (J/cm.sup.2)                                                                              Density*                                     ______________________________________                                        1      0             0           1.114                                        2      5             20          1.337                                        3      5             0           0.559                                        4      2.5           20          1.309                                        5      2.5           0           0.290                                        6      1.25          20          1.242                                        7      1.25          0           0.541                                        ______________________________________                                         *Mean of O.D. values for triplicate sample wells                              Reference:                                                                    Chanh T, Dreesman G, Kanda P, et al. Induction of antiHIV neutralizing        antibodies by synthetic peptides. EMBO J, 5:3065-71, 1986.               

(14) Effect Of Ed6Br (Compound VII) And 420 nm Light On AqueousSuspensions Of H9 Cells

H9 cells (an immortal human T-cell lymphoma) were cultured in RPMI-1640medium containing 10% additional fetal calf serum hold in air with a 5%CO₂ atmosphere at 37.5° C. to a cell density in the range of 10⁵ -10⁷per ml. Cells were, harvested by centrifugation and six 3ml. Aliquotscontaining 1×10⁵ H9 cells per ml suspended in 100% human gerum wereprepared Ed6Br was added to four of the aliquots to give 5 and 25 mMconcentrations, respectively, in each of two samples, and the samplesallowed to set in the dark for 30 minutes. Single samples containing 0,5, and 25 mM Ed6Br concentrations were irradiated with light of 420 # 5nm light filtered from a xenon arc lamp at 20 nW/cm² irradiance to givea light fluence of 10 J/cm². The remaining 3 samples wrapped in aluminumfoil were handled identically but not irradiated. Immediately afterirradiation, the cells in all samples were sedimented by centrifugationat 450 x g for 10 minutes and the cell pellets individually resuspendedto the original 3 ml volume of RPMI-1640 containing an additional 10%fetal calf serum plus Gentamycin antibiotic. The samples were incubatedfor 24 hr. under normal culture conditions cited above. Cell viabilitywas assessed by Trypan Blue dye exclusion counts using a standardhemocytometer after 24 hr incubation. The results of the experiment canbe viewed in Table 20. Ed6Br concentrations of 5 mM or greater clearlyinactivate or kill essentially 100% of H9 cells in suspension whenactivated by light but only approximately 10% or less of the cells wereinactivated by Ed6Br at 25 mM concentration in the absence of light.

                  TABLE 20                                                        ______________________________________                                        Inactivation of H9 Cells (T-Cell Lymphoma)                                    with Ed6Br and 420 nm Light                                                                Live   Dead                                                      Conditions   Cells  Cells.sup.a                                                                            Cells/ml                                                                              % Viable                                 ______________________________________                                        0 μM Ed6Br dark                                                                         114    19       1.14 × 10.sup.6                                                                 85.7                                     0 μM Ed6Br irrad.                                                                       82     11       8.20 × 10.sup.5                                                                 88.2                                     5 μM Ed6Br dark                                                                         77     12       7.70 × 10.sup.5                                                                 86.5                                     5 μM Ed6Br irrad.                                                                        0     47       0.0     0.0                                      25 μM Ed6Br dark                                                                        99     11        9.9 × 10.sup.5                                                                 90.0                                     25 μM Ed6Br irrad.                                                                       0     41       0.0     0.0                                      ______________________________________                                         .sup.a Identified by dark blue color due to Trypan Blue takeup.          

(15) Tissue Bonding Experiments With DiEd6Br (Compound XLIV)

Bonding together of the overlapping surfaces of two strips of swine duramater and of two thin slices of beef muscle with application of DiEd6Brin Cremophor EL as a carrier and irradiation with 420 # 5 nm lightfiltered from a xenon arc lamp were studied.

Strips approximately 3×0.5 cm in dimension were cut from freshlyharvested swine cranial dura mater, washed with 70% ethanol/water andblotted to dryness with standard chemical filter paper. External facesof each member of two pairs of strips were coated with a stock solutionof Cremophor EL saturated at room temperature with DiEd6Br(approximately 2.5 millimolar), overlapped 1 cm with long axes parallel,and clamped between two Pyrex glass slides with edges of theslide-tissue-slide sandwich sealed with Parafilm. One sample wasirradiated with 420 # 5 nm light of 25 mW/cm² irradiance for 20 hrs. atroom temperature to give a total light fluence of approximately 1800J/cm². The companion sample was wrapped in aluminum foil for lightshielding and placed alongside the irradiated sample for the duration ofthe experiment.

Following their respective irradiation or being held in the dark insimilar temperature ambience, one end of a strip of each sample wasclamped and from the other a Styrofoam container was hung by a clamp andfine suspension wires (weight of clamp plus container plus wires=33.3gm). Weights (10 gm each) were added gently in a sequential fashion togive a total weight of 296.3 gm upon which the strip is separated attheir interface within the overlapped region of the irradiated sample.Strips of the un-irradiated sample separated immediately at theirinterface under the 33.3 gm loading of the "weighing" container. Thisresult indicates bonding of the two dura surfaces upon activation ofDiEd6Br with light. Weight loading of the irradiated sample resulted ina uniform elongation and necking pattern of the overlapped regiondemonstrating uniform bonding of the overlapped surfaces. Elongation wasapproximately 50% at shearing of the interface. Upon separation, thestrips rebounded to their nominal original shapes demonstrating noplastic deformation upon weight loading.

Computation of ultimate shear strength at failure (loading force atfailure per overlap area) gave a value of 538 gm/cm² (=1.5×10⁴ Nt/m²)which compares favorably with a value of 286 gm/cm² (=0.8×104 Nt/m²)obtained in thermally bonded human coronary artery immediately aftertreatment as described by Jenkins et al (1988).

Thin strips approximately 10 cm long, 1 cm wide, and 0.05-0.1 cm thickwere sliced from beef steak obtained from a local meat market. Surfacesof the strips of two sample pairs were coated with a stock solution ofDiEd6Br in Cremophor EL (0.1 millimolar concentration) and placedtogether to give approximately 2 cm overlap. One overlapping stripsample was irradiated for 24 hours with water-filtered (IR spectrumabsorber) light from a 150 w xenon arc lamp. Beam diameter incident onthe sample was approximately 3 cm. The other sample was wrapped inaluminum foil, placed alongside the irradiated sample, and served asdark control. Subsequent to irradiation, strips of the dark controlseparated under their own weight when hung vertically whereasdiscernable tactile finger pressure was felt when two hands were used topull the irradiated strip apart, indicating bonding of the strip surfaceupon activation of DiEd6Br with light.

Both dura mater and the the sheaths of the muscle fibers comprising"steak" are connective tissues dominantly comprised of the proteincollagen. The implications of these tissue bonding experiments is thatuse of the 1,8-naphthalimide dyes with activation serves to bondconnective tissues (such as ligament, tendon, cornea of the eye, skin,arterial and venous walls, and duct walls such as of the vas deferens)together via protein crosslinking.

(16) Bleaching And Recovery Of The Fluorescence Of Ed6Br (Compound VII)And DiEd6Br (Compound XLIV)

Part I: Ed6Br Experiment:

A 14.9 millimolar stock solution of Ed6Br in Cremophor EL was used tomake 3 ml of a micellar Cremophor suspension having a concentrationaveraged over the entire sample volume of 100 mM. A 100 mL aliquot wasremoved and diluted with 2.9 ml of distilled water for measurement offluorescence using an SLM 500C spectrofluorometer (excitation, 420 nm,emission, 520 nm). The 3 ml sample was divided into two 1.5 ml aliquotsplaced in quartz crusetts. One sample was irradiated with stirring with420 # 5 nm filtered light from a xenon arc lamp with an irradiance of 20mW/cm² and with the companion sample wrapped in aluminum foil (darkcontrol) and maintained alongside of the irradiated sample. Aliquots of100 mL volume were taken from each sample at times corresponding toillumination exposures of 10, 25, 50, 100, and 200 J/cm² (2 hr.47 min.)and their Ed6Br fluorescence determined immediately. After the entirelight treatment, both the illuminated and dark control samples weredivided into two equal aliquots and held pairwise (dark+previouslyilluminated) at 4° C. and 20° C. in the absence of light. Aliquots of100 mL were taken from each sample after 18 and 43 hr and theirfluorescence emission values determined using the SLM 500C instrumentimmediately after sampling.

The effects of exposure to 420 nm light and subsequent holding of theun-irradiated and previously irradiated samples at 4° C. and 20° C. inthe absence of light can be viewed in Table 21. The steady bleaching ofthe fluorescence emission intensity of Ed6Br during light exposure isevident, whereas the lack of change of the value of fluorescenceemission intensity in the absence of light is also apparent. Theultimate recovery with time of the fluorescence intensity of Ed6Br whileheld at 22° C. and 4° C. are also evident with the rate of recoveryincreasing with temperature. These data indicate that an ultimate valueof approximately 40% of the initial fluorescent intensity is regainedafter irradiation induced loss of about 80% of the fluorescent intensityduring exposure to 200 J/cm².

Part II: DiEd6Br Experiment

In an experiment eventually following the protocol described immediatelyabove in Part I (for Ed6Br), the light-induced bleaching of DiEd6Br with200 J/cm² light exposure and recovery of its fluorescence upon standingin the dark at 22° C. were determined.

The fluorescence decreased to 32.2% of its initial (dark) value uponexposure to 200 J/cm² of 420 nm light and was found to have recovered to62.7 and 64.0% of its initial value after being held for 70 and 166hours at 22° C.

The light induced fluorescence bleaching arises from formation of thenonfluorescent activated g-halocrotonamide species of Ed6Br and DiEd6Brfollowing illumination whereas the regained fluoresence arises fromdeactivation of the activated species not resulting from chemicalreaction. Approximately 50% of the initially activated species recoverin this fashion while the other 50% loose permanently their fluorescenceupon entering into irreversible covalent reaction with a nucleophile,most probably the --OH group of the ricinoleate sidechains of CremophorEL. The presence of the relatively long-lived chemically activeg-halocrotonamide species, a highly efficient alkylating agent, inpreviously light activated Ed6-Br-Cremophor and DiEd6Br-Cremophormixtures suggests use of prior light activation of these1,8-naphthalimide species to generate biologically active species forviral and cellular irradiation.

                  TABLE 21                                                        ______________________________________                                        Bleaching and Recovery of Ed6Br Fluorescence                                  in Aqueous Suspension of Cremophor EL                                         ______________________________________                                        Part A: Fluorescence Bleaching in 420 ± 5 nm Light                         Light Fluence  % Un-irradiated                                                (J/Cm.sup.2)   Fluorescence                                                   ______________________________________                                         0             100                                                            10             97.8                                                           25             96.2                                                           50             74.9                                                           90             48.7                                                           200            18.2                                                           ______________________________________                                        Part B: Fluorescence Recovery in Dark                                         Recovery Time 4° C. Holding                                                                     22° C. Holding                                (Hour)        Temperature                                                                              Temperature                                          ______________________________________                                         0            18.2       18.2                                                 18            24.6       39.4                                                 43            36.8       56.7                                                 ______________________________________                                    

(17) Effects Of 420 nm Radiation On Liposomal-bound Gramacidin D AndDiEd6Br (Compound XLIV)

Two milligram of the hydrophobic peptide Gramacidin D crystals and 8milligrams of L-a-phosphatidyl choline dissolved in chloroform weremixed in a conical 50 ml glass centrifuge tube and evaporated to drynessunder flowing nitrogen gas. Ten ml of phosphate buffered saline (pH=7.3)were added and mixed with a vortex mixer until no lipid layer remainedon tube walls. The lipid suspension, having an average Gramacidinconcentration of 111.1 mM, was then sonicated under nitrogen withcooling via immersion in ice water at a setting of 3 on a Heat SystemsSonicator with microtip. The resulting suspension of phosphatidylcholine liposomes (small unilaminar vesicles) containing Gramacidin Dwas divided into four 2 milliliter aliquots to which DiEd6Br (1millimolar in Cremophor EL) was added to give average dye concentrationof 0, 10, 20, and 50 mM concentration and maintained in the dark for 1hr. at 22° C. to allow partitioning of the dye into the liposomes. Eachof the four 1 milliliter aliquots was divided into 0.5 milliliteraliquots, one for irradiation and one unirradiated control. Irradiationwas, with 420 # 5 nm radiation at an irradiance of 30 mw/cm² to afluence of 100 J/cm².

Following irradiation, both light-exposed and unexposed samples werediluted 1:20 in phosphate buffered saline (pH=7.3) for measurement offluorescence emission intensity of both DiEd6Br and the tryptophanresidues of Gramacidin D. Fluorescence was determined using an SLM 500Cspectrofluorometer. For DiEd6Br excitation and emission wavelengths were425 and 520 nm, respectively, and for the tryptophan residues ofGramacidin D, 290 and 340 nm, respectively.

The decrease in fluorescence emission intensity values of both the dyeand tryptophan with light irradiation can be seen in Table 22. Loss offluorescence intensity reflects the crosslinking to covalent bondingbetween the light activated form DiEd6Br and the nucleophilic tryptophanresidues of Gramacidin D.

                  TABLE 22                                                        ______________________________________                                        Effects on DiEd6Br and Gramacidin D                                           Fluorescences with 420 nm Light Irradiation                                                               Tryptophan                                        Dye Conc. (μM)                                                                         DiEd6Br (ID/DD) %*                                                                            (IT/DT) %**                                       ______________________________________                                        0 μM DiEd6Br                                                                           100             91.23                                             10 μM DiEd6Br                                                                          5.03            10.57                                             20 μM DiEd6Br                                                                          9.24            10.77                                             50 μM DiEd6Br                                                                          20.26           14.40                                             ______________________________________                                         *, **Ratios of irradiated and unirradiated fluorescent omission               intensities of dye (D) and tryptophan (T)                                

(18) Take-Up Of Ed6Br (Compound VII) And DiEd6Br (Compound XLIV) By H9Cells (T-Cell Lymphoma)

H9 cells (an immortal human T-cell lymphoma) were cultured in RPMI-1640medium containing 10% additional fetal calf serum held in air with a 5%CO₂ atmosphere at 37.5° C. to a cell density in the range of 10⁵ -10⁷per ml. Cells were harvested by centrifugation and four 3-milliliteraliquots containing 5×10⁵ H9 cells per ml suspended in RPM I-1640 mediumwith 10% additional fetal calf serum were prepared. Ed6Br was added tothree aliquots to give 5, and 25 mM concentrations, respectively. Thefourth aliquot served as a dye-free control. Immediately after additionof the dyes, 1 ml samples were harvested from each aliquot and the cellswashed by dilution into 3 ml phosphate buffered saline (pH 7.3),followed by two repetitions of centrifugation, decantation of thesupernatant, and resuspension of the cell pellet in the buffered salinesolution, and a final centrifugation, decantation, and retention of thecell pellet at 4° C. for subsequent analysis. At the subsequent elapsedincubation times at 1, 2, 3, and 4 hrs., one milliliter samples wereharvested and treated in an identical fashion. After treatment of the4-hr. sample, the cells of each sample were lysed in 3 ml of an 0.1%solution of ctyltetraammonium bromide detergent in phosphate bufferedsaline (pH=7.3). The fluorescence emission at 520 nm (420 nm excitation)of each sample was determined with a SLM SPT 500C spectrofluorometer.Additionally, the emission intensity of samples of these detergentsolutions having, respectively, dye concentrations of 0, 0.1, 0.2, 0.5,0.8, 1.0, 1.2, 1.5, 2, 3, 4, and 5 mM were prepared as standards forcalibrating the concentration dependence of the fluorescence emission.These data were used with the fluorescence emission intensity valuesobtained from the cell samples to determine the total concentration ofdye taken up by the 5×10⁵ ml H9 cells in each cell aliquot, and then theaverage quantity of dye sequestered per cell at the various exposuretimes and external dye concentrations was computed.

The results can be viewed in Table 23. The increase and saturation inamount of Ed6Br taken-up with increasing exposure time and the increasewith external dye concentration are readily discerned. Using an averagediameter of 12 micron for the H9 cell determined in our laboratory usinglight microscopy, the average dye concentration in the H9 cell can beestimated from the total number of moles taken-up per cell. For example,a 4 hr exposure to an external Ed6Br concentration of 25 mM resulted intake-up of Ed6Br by the average H9 cell to result a concentration ofabout 1.92 millimolar, approximately 77 times greater than the externalconcentration. Similar calculation of average cellular concentrationafter four hr exposure for exposure to 1 and 5 mm external concentrationof Ed6Br resulted also in approximately a 70-fold increasedconcentration of the dye by the cell.

                  TABLE 23                                                        ______________________________________                                        Time Dependent Up-take of Ed6Br by                                            H9 Cells (T-Cell Lymphoma at 22° C.)                                   Exposure Time                                                                              External Ed6BR                                                                              Cellular Ed6Br                                     (hr.)        Concentration (μM)                                                                       (a° mol/cell).sup.(a)                       ______________________________________                                        0            1              91.2                                              1                          130.2                                              2                          189.8                                              3                          186.0                                              4                          208.3                                              0            5             165.6                                              1                          385.1                                              2                          437.2                                              3                          468.8                                              4                          548.9                                              0            25            316.2                                              1                          716.3                                              2                          1073.4                                             3                          1689.3                                             4                          1748.8                                             ______________________________________                                         .sup.(a) 1 a° mol = 1 × 10.sup.-18 mol                      

(19) Effects On H9 Cells (T-cell Lymphoma) Of Ed6Br (Compound VII) inCremophor EL With Prior Activation By 420 #5 nm Light

H9 cells were cultivated in RPMI-1640 medium containing an additional10% fetal calf serum at 37.5° C. in air having added 5% CO₂. A 5.46 mlaliquot of cell suspension containing 9.16×10⁵ H9 cells per ml washarvested, the cells sedimented at 450 x g for 10 minutes andresuspended in fresh growth medium. Two hundred microliter aliquots ofthe suspension were centrifuged as above and the resulting cell pelletserved for resuspension in dye plus medium suspension for this study.

Six milliliters of suspension of Cremophor EL micelles containing Ed6Brin RPMI-1640 containing 10% additional fetal calf serum was made bydiluting a 2.5 millimeter solution of the dye in Cremophor EL to give anaverage Ed6Br concentration of 100 mM, and the resulting suspensiondivided into 2 three milliliter aliquots. One aliquot was irradiatedwith 420 #5 nm light filtered from a xenon arc lamp at an irradiance of20 mW/cm² to a total fluence of 200 J/cm². The companion aliquot waswrapped in aluminum foil and handled in identical fashion to theirradiated sample. These two aliquots served as stocks for deformingeffects of both previously irradiated and un-irradiated Ed6Br atdifferent concentrations from 0 up to 100 mM on H9 cells kept in thedark. Dulutions of the stock dye suspension into additional fresh growthmedium were made within 1 hour of light irradiation to give dye-mediumsuspensions with average Ed6Br concentrations of 0, 1, 10, 25, 50, 75,and 200 mM for both previously irradiated and dark dye samples. The H9cell pellets were resuspended in these dye-Cremophor EL suspensions andheld at 37.5° C. in an air atmosphere containing an additional 5% CO₂.After 24 hrs. culture of the number of total and viable H0 cells wasdetermined by Trypan Blue exclusion using a hemocytometer and microscopefor cell counting. The number of killed cells was determined as thoseexhibiting the blue dye color. See Table 24.

                  TABLE 24                                                        ______________________________________                                        Inactivation of H9 Cells (T-Cell Lymphoma) with                               Ed6BR Previously Activated with 420 nm Light                                  Ed6BR                                                                         Concentration Previous Light                                                  (μM)       Activation  % Viable Cells                                      ______________________________________                                         0            No          72.6                                                 1            No          72.7                                                 1            Yes         68.4                                                10            No          77.2                                                10            Yes         31.8                                                25            No          57.9                                                25            Yes         4.9                                                 50            No          27.4                                                50            Yes         0.0                                                 75            No          33.9                                                75            Yes         0.0                                                 100           No          28.0                                                100           Yes         0.0                                                 ______________________________________                                    

While the present invention has been particularly described in terms ofspecific embodiments thereof, it will be understood in view of thepresent disclosure that numerous variations upon the invention are nowenabled to those skilled in the art, which variations yet reside withinthe scope of the present invention. Accordingly, the invention is to bebroadly construed, and limited only by the scope and spirit of theclaims now appended hereto.

What is claimed is:
 1. A non-azo N-substituted-1,8-naphthalimidecompound substituted by, at a 3-position, a nucleofuge and, at a4-position, a heteroatomic electron-releasing group, aid heteroatomicelectron-releasing group being characterized as having a heteroatomdirectly linked to said 4-position and having at least one hydrogendirectly attached to said heteroatom, said non-azoN-substituted-1,8-naphthalimide compound being characterized aspredominantly hydrophobic and, after being activated by a sufficientamount of activating agent gives an activated derivative.
 2. Thecompound of claim 1, wherein said nucleofuge is a halogen, a sulfonateester, or a nitrogen-containing leaving group.
 3. The compound of claim1, wherein said heteroatom is nitrogen or oxygen.
 4. The compound ofclaim 1, wherein said activating agent is light energy, X-ray radiation,or thermalized neutron particles.
 5. The compound of claim 1, whereinsaid activating agent is light energy having a wavelength range of fromabout 200 nm to about 1000 nm.
 6. The compound of claim 5, wherein saidlight energy has a wavelength range of from about 290 nm to about 750nm.
 7. The compound of claim 5, wherein said light energy has awavelength range of from about 600 nm to about 750 nm.
 8. The compoundof claim 5, wherein said light energy has a wavelength range of fromabout 650 nm to about 700 nm.
 9. The compound of claim 1, wherein saidactivated derivative is characterized as being relatively non-toxic tonormal tissues or normal cells at effective dosages yet can undergophotoinduced tautomeric alkylation with a target protein- basednucleophile.
 10. A naphthalimide compound having the formula ##STR1##which is a mixture of stereoisomers wherein: R and R':saturatedstraight-chain or branched-chain alkyl, C_(n) H_(2n+1) (n=1-30);partially or totally fluorinated saturated straight-chain orbranched-chain alkyl, C_(n) H_(q) F_(2n-q+1) (n=1-30, 0≦q≦2n);unsaturated straight-chain or branched-chain alkyl, C_(n) H_(2m+1)(n=1-30, 1≦m<n); partially or totally fluorinated unsaturatedstraight-chain or branched-chain alkyl, C_(n) H_(q) F_(2m-q+1) (n=1-30,1≦m<n, 0≦q≦2m); alicyclic (monocyclic or polycyclic, fused-ring,bridged-ring or spirocyclic) alkyl, C_(n) H_(2m+1) (n=1-30, 1≦m≦n) withsaturated or unsaturated side-chains (branched or unbranched); partiallyor totally fluorinated alicyclic (monocyclic or polycyclic, fused-ring,bridged-ring or spirocyclic) alkyl, C_(n) H_(q) F_(2m-q+1) (n=1-30,1≦m<n, 0≦q≦2m) with saturated or unsaturated side-chains (branched orunbranched); aryl substituted branched, unbranched or alicyclic,saturated or unsaturated alkyl, ArC_(n) H_(2m+1) (n=1-30, 1≦m≦n), whereAr is an aromatic moiety (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole or their substituted or benzo derivatives) located eitherwithin the chain of the alkyl group or at a terminus; partially ortotally fluorinated aryl substituted branched, unbranched or alicyclic,saturated or unsaturated alkyl, ArC_(n) H_(q) F_(2m-q+1) (n=1-30, 1≦m≦n,0≦q≦2m), where Ar is an aromatic moiety (benzene, naphthalene, azulene,phenanthrene, anthracene, pyridine, quinoline, isoquinoline, purine,pyrimidine, pyrrole, indole, carbazole, furan, thiophene, imidazole,isoxazole, thiazole or their substituted or benzo derivatives) locatedeither within the chain of the alkyl group or at a terminus; substitutedsaturated straight-chain or branched-chain alkyl, C_(n) H_(2n-p+1) Y_(p)(n=1-30, 1≦p≦10) where Y is a substituted aryl group (benzene,naphthalene, azulene, phenanthrene, anthracene, pyridine, quinoline,isoquinoline, purine, pyrimidine, pyrrole, indole, carbazole, furan,thiophene, imidazole, isoxazole, thiazole or their substituted or benzoderivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, or ROPO₂ ²⁻), asilicon derivative (SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, orhalo), or a boron derivative (BZ₂, where Z is alkyl, aryl, alkoxy,aryloxy, or halo); substituted partially or totally fluorinatedstraight-chain or branched-chain alkyl, C_(n) H_(q) F_(2n-p-q+1) Y_(p)(n=1-30, 1≦p≦10 0≦q≦2n); substituted unsaturated straight-chain orbranched-chain alkyl, C_(n) H_(2m-p+1) Y_(p) (n=1-30, 1≦m<n, 1≦p≦10)where Y is a substituted aryl group (benzene, naphthalene, azulene,phenanthrene, anthracene, pyridine, quinoline, isoquinoline, purine,pyrimidine, pyrrole, indole, carbazole, furan, thiophene, imidazole,isoxazole, thiazole or their substituted or benzo derivatives), acharged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, or ROPO₂ ²⁻), a silicon derivative(SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, or halo), or a boronderivative (BZ₂, where Z is alkyl, aryl, alkoxy, aryloxy, or halo);substituted partially or totally fluorinated straight-chain orbranched-chain alkyl, C_(n) H_(q) F_(2m-p-q+1) Y_(p) (n=1-30, 1≦m<n,1≦p≦10, 0≦q≦2m); substituted alicyclic (monocyclic or polycyclic,fused-ring, bridged-ring or spirocyclic) alkyl, C_(n) H_(2m+1) (n=1-30,1≦m≦n) with saturated or unsaturated side-chains (branched orunbranched) where Y is a substituted aryl group (benzene, naphthalene,azulene, phenanthrene, anthracene, pyridine, quinoline, isoquinoline,purine, pyrimidine, pyrrole, indole, carbazole, furan, thiophene,imidazole, isoxazole, thiazole or their substituted or benzoderivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, or ROPO₂ ²⁻), asilicon derivative (SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, orhalo), or a boron derivative (BZ₂, where Z is alkyl, aryl, alkoxy,aryloxy, or halo); substituted partially or totally fluorinatedalicyclic (monocyclic or polycyclic, fused-ring, bridged-ring orspirocyclic) alkyl, C_(n) H_(q) F_(2m-p-q+1) Y_(p) (n=1-30, 1≦m<n,1≦p≦10, 00≦q≦2m) with saturated or unsaturated sidechains (branched orunbranched) where Y is a substituted aryl group (benzene, naphthalene,azulene, phenanthrene, anthracene, pyridine, quinoline, isoquinoline,purine, pyrimidine, pyrrole, indole, carbazole, furan, thiophene,imidazole, isoxazole, thiazole or their substituted or benzoderivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, or ROPO₂ ²⁻), asilicon derivative (SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, orhalo), or a boron derivative (BZ₂, where Z is alkyl, aryl, alkoxy,aryloxy, or halo); substituted aryl substituted branched, unbranched oralicyclic, saturated or unsaturated alkyl, ArC_(n) H_(2m-p+1) Y_(p)(n=1-30, 1≦m≦n, 1≦p≦10), where Ar is an aromatic moiety (benzene,naphthalene, azulene, phenanthrene, anthracene, pyridine, quinoline,isoquinoline, purine, pyrimidine, pyrrole, indole, carbazole, furan,thiophene, imidazole, isoxazole, thiazole or their substituted or benzoderivatives) located either within the chain of the alkyl group or at aterminus, and where Y is a substituted aryl group (defined above), acharged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, or ROPO₂ ⁻), a silicon derivative(SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, or halo), or a boronderivative (BZ₂, where Z is alkyl, aryl, alkoxy, aryloxy, or halo);substituted partially or totally fluorinated aryl substituted branched,unbranched or alicyclic, saturated or unsaturated alkyl, ArC_(n) H_(q)F_(2m-p-q+1) Y_(p) (n=1-30, 1≦m≦n, 1≦p≦10, 0≦q≦2m), where Ar is anaromatic moiety (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole or their substituted or benzo derivatives) located eitherwithin the chain of the alkyl group or at a terminus, and where Y is asubstituted aryl group (defined above), a charged group (CO₂ ⁻, SO₃ ⁻,PO₃ ²⁻, or ROPO₂ ²⁻), a silicon derivative (SiZ₃, where Z is alkyl,aryl, alkoxy, aryloxy, or halo), or a boron derivative (BZ₂, where Z isalkyl, aryl, alkoxy, aryloxy, or halo); a straight-chain orbranched-chain group capable of complexing a metal ion, C_(n) H_(2m+1)Y_(q) (n=1-30, 1≦q≦n, 1≦q≦n/2), where Y is O, S, Se, NH, N-R, N-Ar, PH,P-R and/or P-Ar and their acyl (including aminoacyl or peptide)derivatives, and where R is saturated alkyl (C_(n) H_(2n+1), n=1-30),fluorinated saturated alkyl (C_(n) H_(q) F_(2n-q+1), n=1-30, 0≦q≦2n),unsaturated alkyl (C_(n) H_(2m+1), n=1-30, 1≦m<n), fluorinatedunsaturated alkyl (C_(n) H_(q) F_(2m-q+1), n=1-30, 1≦m≦n, 0≦q≦2m),alicyclic (C_(n) H_(2m+1), n=1-30, 1<m≦n), fluorinated alicyclic (C_(n)H_(q) F_(2m-q+1), n=1-30, 1≦m<n, 0≦q≦2m), and where Ar is an aromaticmoiety (benzene, naphthalene, azulene, phenanthrene, anthracene,pyridine, quinoline, isoquinoline, purine, pyrimidine, pyrrole, indole,carbazole, furan, thiophene, imidazole, isoxazole, thiazole or theirsubstituted or benzo derivatives) located either within the chain of thealkyl group or at a terminus; a substituted alicyclic group capable ofcomplexing a metal ion, C_(n) H_(2m+1) Y_(q) (n=1-30, 1≦q≦n, 1≦q≦n/2),where Y is O, S, Se, NH, N-R, N-Ar, PH, P-R and/or P-Ar or their acyl(including aminoacyl or peptide) derivatives, and where R is saturatedalkyl (C_(n) H_(2n+1), n=1-30), fluorinated saturated alkyl (C_(n) H_(q)F_(2n-q+1), n=1-30, 0≦q≦2n), unsaturated alkyl (C_(n) H_(2m+1), n=1-30,1≦m<n), fluorinated unsaturated alkyl (C_(n) H_(q) F_(2m-q+1), n=1-30,1≦m<n, 0≦q≦2m), alicyclic (C_(n) H_(2m+1), n=1-30, 1≦m≦n), fluorinatedalicyclic (C_(n) H_(q) F_(2m-q+1), n=1-30, 1≦m<n, 0≦q≦2m), and where Aris an aromatic moiety (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole or their substituted or benzo derivatives) located eitherwithin the chain of the alkyl group or at a terminus; or a modified orunmodified biomolecule (steroids, phospholipids, mono-, di- ortriglycerides, mono- or polysaccharides, nucleosides, and polypeptides),where Y is a substituted aryl group (defined above), a charged group(CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, and ROPO₂ ²⁻), a silicon derivative (SiZ₃, whereZ is alkyl, aryl, alkoxy, aryloxy, or halo), or a boron derivative (BZ₂,where Z is alkyl, aryl, alkoxy, aryloxy, or halo), or a biocompatibleoligomer or polymer (polyglycolic acid);X: halogen (F, Cl, Br, I):sulfonate ester (alkanesulfonates, partially or totally fluorinatedalkanesulfonates, arenesulfonates, or partially or totally fluorinatedarenesulfonates): or a nitrogen leaving group (diazonium ion);orpharmaceutically acceptable salts thereof.
 11. A non-azoN-substituted-1,8-naphthalimide compound having at least two1,8-naphthalimide moieties each substituted by, at a 3-position, anucleofuge and, at a 4-position, a heteroatomic electron-releasinggroup, said heteroatomic electron-releasing group being characterized ashaving a heteroatom directly linked to said 4-position and having atleast one hydrogen directly attached to said heteroatom, said non-azoN-substituted-1,8-naphthalimide compound being characterized aspredominantly hydrophobic and, after being activated by a sufficientamount of activating agent, gives an activated derivative.
 12. Thecompound of claim 11 wherein said nucleofuge is a halogen, a sulfonateester, or a nitrogen-containing leaving group.
 13. The compound or claim11, wherein said heteroatom is nitrogen or oxygen.
 14. The compound ofclaim 11, wherein said activating agent is light energy, X-rayradiation, or thermalized neutron particles.
 15. The compound of claim11, wherein said activating agent is light energy having a wavelengthrange of from about 200 nm to about 1000 nm.
 16. The compound of claim15, wherein said light energy has a wavelength range of from about 290nm to about 750 nm.
 17. The compound of claim 15, wherein said lightenergy has a wavelength range of from about 600 nm to about 750 nm. 18.The compound of claim 15, wherein said light energy has a wavelengthrange of from about 650 nm to about 700 nm.
 19. The compound of claim11, wherein said activated derivative is characterized as beingrelatively non-toxic to normal tissues or normal cells at effectivedosages yet can undergo photoinduced tautomeric alkylation with a targetprotein-based nucleophile.
 20. A bis-naphthalimide compound having theformula ##STR2## which is a mixture of stereoisomers wherein: R andR':saturated straight-chain or branched-chain alkyl, C_(n) H_(2n+1)(n=1-30); partially or totally fluorinated saturated straight-chain orbranched-chain alkyl, C_(n) H_(q) F_(2n-q+1) (n=1-30, 0≦q≦2n);unsaturated straight-chain or branched-chain alkyl, C_(n) H_(2m+1)(n=1-30, 1≦m<n); partially or totally fluorinated unsaturatedstraight-chain or branched-chain alkyl, C_(n) H_(q) F_(2m-q+1) (n=1-30,1≦m<n, 0≦q≦2m); alicyclic (monocyclic or polycyclic, fused-ring,bridged-ring or spirocyclic) alkyl, C_(n) H_(2m+1) (n=1-30, 1≦m≦n) withsaturated or unsaturated side-chains (branched or unbranched); partiallyor totally fluorinated alicyclic (monocyclic or polycyclic, fused-ring,bridged-ring or spirocyclic) alkyl, C_(n) H_(q) F_(2m-q+1) (n=1-30,1≦m<n, 0≦m≦2m) with saturated or unsaturated side-chains (branched orunbranched); aryl substituted branched, unbranched or alicyclic,saturated or unsaturated alkyl, ArC_(n) H_(2m+1) (n=1-30, 1≦m≦n), whereAr is an aromatic moiety (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole or their substituted or benzo derivatives) located eitherwithin the chain of the alkyl group or at a terminus; partially ortotally fluorinated aryl substituted branched, unbranched or alicyclic,saturated or unsaturated alkyl, ArC_(n) H_(q) F_(2m-q+1) (n=1-30, 1≦m≦n,0≦q≦2m), where Ar is an aromatic moiety (benzene, naphthalene, azulene,phenanthrene, anthracene, pyridine, quinoline, isoquinoline, purine,pyrimidine, pyrrole, indole, carbazole, furan, thiophene, imidazole,isoxazole, thiazole or their substituted or benzo derivatives) locatedeither within the chain of the alkyl group or at a terminus; substitutedsaturated straight-chain or branched-chain alkyl, C_(n) H_(2n-p+1) Y_(p)(n=1-30, 1≦p≦10) where Y is a substituted aryl group (benzene,naphthalene, azulene, phenanthrene, anthracene, pyridine, quinoline,isoquinoline, purine, pyrimidine, pyrrole, indole, carbazole, furan,thiophene, imidazole, isoxazole, thiazole or their substituted or benzoderivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, or ROPO₂ ²⁻), asilicon derivative (SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, orhalo), or a boron derivative (BZ₂, where Z is alkyl, aryl, alkoxy,aryloxy, or halo); substituted partially or totally fluorinatedstraight-chain or branched-chain alkyl, C_(n) H_(q) F_(2n-p-q+1) Y_(p)(n=1-30, 1≦p≦10 0≦q≦2n); substituted unsaturated straight-chain orbranched-chain alkyl, C_(n) H_(2m-p+1) Y_(p) (n=1-30, 1≦m<n, 1≦c≦10)where Y is a substituted aryl group (benzene, naphthalene, azulene,phenanthrene, anthracene, pyridine, quinoline, isoquinoline, purine,pyrimidine, pyrrole, indole, carbazole, furan, thiophene, imidazole,isoxazole, thiazole or their substituted or benzo derivatives), acharged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, or ROPO₂ ²⁻), a silicon derivative(SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, or halo), or a boronderivative (BZ₂, where Z is alkyl, aryl, alkoxy, aryloxy, or halo);substituted partially or totally fluorinated straight-chain orbranched-chain alkyl, C_(n) H_(q) F_(2m-p-q+1) Y_(p) (n=1-30, 1≦m<n,1≦p≦10, 0≦q≦2m); substituted alicyclic (monocyclic or polycyclic,fused-ring, bridged-ring or spirocyclic) alkyl, C_(n) H_(2m+1) (n=1-30,1≦m≦n) with saturated or unsaturated side-chains (branched orunbranched) where Y is a substituted aryl group (benzene, naphthalene,azulene, phenanthrene, anthracene, pyridine, quinoline, isoquinoline,purine, pyrimidine, pyrrole, indole, carbazole, furan, thiophene,imidazole, isoxazole, thiazole or their substituted or benzoderivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, or ROPO₂ ²⁻), asilicon derivative (SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, orhalo), or a boron derivative (BZ₂, where Z is alkyl, aryl, alkoxy,aryloxy, or halo); substituted partially or totally fluorinatedalicyclic (monocyclic or polycyclic, fused-ring, bridged-ring orspirocyclic) alkyl, C_(n) H_(q) F_(2m-p-q+1) Y_(p) (n=1-30, 1≦m<n,1≦p≦10, 0≦q≦2m) with saturated or unsaturated side-chains (branched orunbranched) where Y is a substituted aryl group (benzene, naphthalene,azulene, phenanthrene, anthracene, pyridine, quinoline, isoquinoline,purine, pyrimidine, pyrrole, indole, carbazole, furan, thiophene,imidazole, isoxazole, thiazole or their substituted or benzoderivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, or ROPO₂ ²⁻), asilicon derivative (SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, orhalo), or a boron derivative (BZ₂, where Z is alkyl, aryl, alkoxy,aryloxy, or halo); substituted aryl substituted branched, unbranched oralicyclic, saturated or unsaturated alkyl, ArC_(n) H_(2m-p+1) Y_(p)(n=1-30, 1≦m≦n, 1≦p≦10), where Ar is an aromatic moiety (benzene,naphthalene, azulene, phenanthrene, anthracene, pyridine, quinoline,isoquinoline, purine, pyrimidine, pyrrole, indole, carbazole, furan,thiophene, imidazole, isoxazole, thiazole or their substituted or benzoderivatives) located either within the chain of the alkyl group or at aterminus, and where Y is a substituted aryl group (defined above), acharged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, or ROPO₂ ²⁻), a silicon derivative(SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, or halo), or a boronderivative (BZ₂, where Z is alkyl, aryl, alkoxy, aryloxy, or halo);substituted partially or totally fluorinated aryl substituted branched,unbranched or alicyclic, saturated or unsaturated alkyl, ArC_(n) H_(q)F_(2m-p-q+1) Y_(p) (n=1-30, 1≦m≦n, 1≦p≦10, 0≦q≦2m), where Ar is anaromatic moiety (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole or their substituted or benzo derivatives) located eitherwithin the chain of the alkyl group or at a terminus, and where Y is asubstituted aryl group (defined above), a charged group (CO₂ ⁻, SO₃ ⁻,PO₃ ²⁻, or ROPO₂ ²⁻), a silicon derivative (SiZ₃, where Z is alkyl,aryl, alkoxy, aryloxy, or halo), or a boron derivative (BZ₂, where Z isalkyl, aryl, alkoxy, aryloxy, or halo); a straight-chain orbranched-chain group capable of complexing a metal ion, C_(n) H_(2m+1)Y_(q) (n=1-30, 1≦q≦n, 1≦q≦n/2), where Y is O, S, Se, NH, N-R, N-Ar, PH,P-R and/or P-Ar or their acyl (including aminoacyl or peptide)derivatives, and where R is saturated alkyl (C_(n) H_(2n+1), n=1-30),fluorinated saturated alkyl (C_(n) H_(q) F_(2n-q+1), n=1-30, 0≦q≦2n),unsaturated alkyl (C_(n) H_(2m+1), n=1-30, 1≦m<n), fluorinatedunsaturated alkyl (C_(n) H_(q) F_(2m-q+1), n=1-30, 1≦m<n, 0≦q≦2m),alicyclic (C_(n) H_(2m+1), n=1-30, 1≦m≦n), fluorinated alicyclic (C_(n)H_(q) F_(2m-q+1), n=1-30, 1≦m<n, 0≦q≦2m), and where Ar is an aromaticmoiety (benzene, naphthalene, azulene, phenanthrene, anthracene,pyridine, quinoline, isoquinoline, purine, pyrimidine, pyrrole, indole,carbazole, furan, thiophene, imidazole, isoxazole, thiazole or theirsubstituted or benzo derivatives) located either within the chain of thealkyl group or at a terminus; a substituted alicyclic group capable ofcomplexing a metal ion, C_(n) H_(2m+1) Y_(q) (n=1-30, 1≦q≦n, 1≦q≦n/2),where Y is O, S, Se, NH, N-R, N-Ar, PH, P-R and/or P-Ar or their acyl(including aminoacyl or peptide) derivatives, and where R is saturatedalkyl (C_(n) H_(2n+1), n=1-30), fluorinated saturated alkyl (C_(n) H_(q)F_(2n-q+1), n=1-30, 0≦q≦2n), unsaturated alkyl (C_(n) H_(2m+1), n=1-30,1≦m<n), fluorinated unsaturated alkyl (C_(n) H_(q) F_(2m-q+1), n=1-30,1≦m<n, 0≦q≦2m), alicyclic (C_(n) H_(2m+1), n=1-30, 1≦m≦n), fluorinatedalicyclic (C_(n) H_(q) F_(2m-q+1), n=1-30, 1≦m<n, 0≦q≦2m), and where Aris an aromatic moiety (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole or their substituted or benzo derivatives) located eitherwithin the chain of the alkyl group or at a terminus; or a modified orunmodified biomolecule (steroids, phospholipids, mono-, di- ortriglycerides, mono- and polysaccharides, nucleosides, or polypeptides),where Y is a substituted aryl group (defined above), a charged group(CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, and ROPO₂ ²⁻), a silicon derivative (SiZ₃, whereZ is alkyl, aryl, alkoxy, aryloxy, or halo), or a boron derivative (BZ₂,where Z is alkyl, aryl, alkoxy, aryloxy, or halo), or a biocompatibleoligomer or polymer (polyglycolic acid);X: halogen (F, Cl, Br, I);sulfonate ester (alkanesulfonates, partially or totally fluorinatedalkanesulfonates, arenesulfonates, or partially or totally fluorinatedarenesulfonates); or a nitrogen leaving group (diazonium ion); saturatedstraight-chain or branched-chain alkyl, C_(n) H_(2n) (n=1-30); partiallyor totally fluorinated saturated straight-chain or branched-chain alkyl,C_(n) H_(q) F_(2n-q) (n=1-30, 0≦q≦2n); unsaturated straight-chain orbranched-chain alkyl, C_(n) H_(2m) (n=1-30, 1≦m<n); partially or totallyfluorinated unsaturated straight-chain or branched-chain alkyl, C_(n)H_(q) F_(2m-q) (n=1-30, 1≦m<n, 0≦q≦2m); alicyclic (monocyclic orpolycyclic, fused-ring, bridged-ring or spirocyclic) alkyl, C_(n) H_(2m)(n=1-30, 1≦m≦n) with saturated or unsaturated side-chains (branched orunbranched); partially or totally fluorinated alicyclic (monocyclic orpolycyclic, fused-ring, bridged-ring or spirocyclic) alkyl, C_(n) H_(q)F_(2m-q) (n=1-30, 1≦m<n, 0≦q≦2m) with saturated or unsaturatedside-chains (branched or unbranched); aryl substituted branched,unbranched or alicyclic, saturated or unsaturated alkyl, ArC_(n) H_(2m)(n=1-30, 1≦m≦n), where Ar is an aromatic moiety (benzene, naphthalene,azulene, phenanthrene, anthracene, pyridine, quinoline, isoquinoline,purine, pyrimidine, pyrrole, indole, carbazole, furan, thiophene,imidazole, isoxazole, thiazole or their substituted or benzoderivatives) located either within the chain of the alkyl group or at aterminus; partially or totally fluorinated aryl substituted branched,unbranched or alicyclic, saturated or unsaturated alkyl, ArC_(n) H_(q)F_(2m-q) (n=1-30, 1≦m≦n, 0≦q≦2m), where Ar is an aromatic moiety(benzene, naphthalene, azulene, phenanthrene, anthracene, pyridine,quinoline, isoquinoline, purine, pyrimidine, pyrrole, indole, carbazole,furan, thiophene, imidazole, isoxazole, thiazole or their substituted orbenzo derivatives) located either within the chain of the alkyl group orat a terminus; substituted saturated straight-chain or branched-chainalkyl, C_(n) H_(2n-p) Y_(p) (n=1-30, 1≦p≦10) where Y is a substitutedaryl group (benzene, naphthalene, azulene, phenanthrene, anthracene,pyridine, quinoline, isoquinoline, purine, pyrimidine, pyrrole, indole,carbazole, furan, thiophene, imidazole, isoxazole, thiazole or theirsubstituted or benzo derivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₃²⁻, or ROPO₂ ²⁻), a silicon derivative (SiZ₃, where Z is alkyl, aryl,alkoxy, aryloxy, or halo), or a boron derivative (BZ₂, where Z is alkyl,aryl, alkoxy, aryloxy, or halo); substituted partially or totallyfluorinated straight-chain or branched-chain alkyl, C_(n) H_(q)F_(2n-p-q) Y_(p) (n=1-30, 1≦p≦10 0≦q≦2n); substituted unsaturatedstraight-chain or branched-chain alkyl, C_(n) H_(2m-p) Y_(p) (n=1-30,1≦m<n, 1≦p≦10) where Y is a substituted aryl group (benzene,naphthalene, azulene, phenanthrene, anthracene, pyridine, quinoline,isoquinoline, purine, pyrimidine, pyrrole, indole, carbazole, furan,thiophene, imidazole, isoxazole, thiazole or their substituted or benzoderivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, or ROPO₂ ²⁻), asilicon derivative (SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, orhalo), or a boron derivative (BZ₂, where Z is alkyl, aryl, alkoxy,aryloxy, or halo); substituted partially or totally fluorinatedstraight-chain or branched-chain alkyl, C_(n) H_(q) F_(2m-p-q) Y_(p)(n=1-30, 1≦m<n, 1≦p≦10, 0≦q≦2m); substituted alicyclic (monocyclic orpolycyclic, fused-ring, bridged-ring or spirocyclic) alkyl, C_(n) H_(2m)(n=1-30, 1≦m≦n) with saturated or unsaturated side-chains (branched orunbranched) where Y is a substituted aryl group (benzene, naphthalene,azulene, phenanthrene, anthracene, pyridine, quinoline, isoquinoline,purine, pyrimidine, pyrrole, indole, carbazole, furan, thiophene,imidazole, isoxazole, thiazole or their substituted or benzoderivatives), a charged group (CO₂ ⁻¹, SO₃ ⁻, PO₃ ²⁻, or ROPO₂ ²⁻), asilicon derivative (SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, orhalo), or a boron derivative (BZ₂, where Z is alkyl, aryl, alkoxy,aryloxy, or halo); substituted partially or totally fluorinatedalicyclic (monocyclic or polycyclic, fused-ring, bridged-ring orspirocyclic) alkyl, C_(n) H_(q) F_(2m-p-q) Y_(p) (n=1-30, 1≦m<n, 1≦p≦10,0≦q≦2m) with saturated or unsaturated side-chains (branched orunbranched) where Y is a substituted aryl group (benzene, naphthalene,azulene, phenanthrene, anthracene, pyridine, quinoline, isoquinoline,purine, pyrimidine, pyrrole, indole, carbazole, furan, thiophene,imidazole, isoxazole, thiazole or their substituted or benzoderivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, or ROPO₂ ²⁻), asilicon derivative (SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, orhalo), or a boron derivative (BZ₂, where Z is alkyl, aryl, alkoxy,aryloxy, or halo); substituted aryl substituted branched, unbranched oralicyclic, saturated or unsaturated alkyl, ArC_(n) H_(2m-p) Y_(p)(n=1-30, 1≦m≦n, 1≦p≦10), where Ar is an aromatic moiety (benzene,naphthalene, azulene, phenanthrene, anthracene, pyridine, quinoline,isoquinoline, purine, pyrimidine, pyrrole, indole, carbazole, furan,thiophene, imidazole, isoxazole, thiazole or their substituted or benzoderivatives) located either within the chain of the alkyl group or at aterminus, and where Y is a substituted aryl group (defined above), acharged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, or ROPO₂ ²⁻), a silicon derivative(SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, or halo), or a boronderivative (BZ₂, where Z is alkyl, aryl, alkoxy, aryloxy, or halo);substituted partially or totally fluorinated aryl substituted branched,unbranched or alicyclic, saturated or unsaturated alkyl, ArC_(n) H_(q)F_(2m-p-q) Y_(p) (n=1-30, 1≦m≦n, 1≦p≦10, 0≦q≦2m), where Ar is anaromatic moiety (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole or their substituted or benzo derivatives) located eitherwithin the chain of the alkyl group or at a terminus, and where Y is asubstituted aryl group (defined above), a charged group (CO₂ ⁻, SO₃ ⁻,PO₃ ²⁻, or ROPO₂ ²⁻), a silicon derivative (SiZ₃, where Z is alkyl,aryl, alkoxy, aryloxy, or halo), or a boron derivative (BZ₂, where Z isalkyl, aryl, alkoxy, aryloxy, or halo); a straight-chain orbranched-chain group capable of complexing a metal ion, C_(n) H_(2m)Y_(q) (n=1-30, 1≦q≦n, 1≦q≦n/2), where Y is O, S, Se, NH, N-R, N-Ar, PH,P-R and/or P-Ar and their acyl (including aminoacyl and peptide)derivatives, and where R is saturated alkyl (C_(n) H_(2n), n=1-30),fluorinated saturated alkyl (C_(n) H_(q) F_(2n-q), n=1-30, 0≦q≦2n),unsaturated alkyl (C_(n) H_(2m), n=1-30, 1≦m<n), fluorinated unsaturatedalkyl (C_(n) H_(q) F_(2m-q), n=1-30, 1≦m<n, 0≦q≦2m), alicyclic (C_(n)H_(2m), n=1-30, 1≦m≦n), fluorinated alicyclic (C_(n) H_(q) F_(2m-q),n=1-30, 1≦m<n, 0≦q≦2m), and where Ar is an aromatic moiety (benzene,naphthalene, azulene, phenanthrene, anthracene, pyridine, quinoline,isoquinoline, purine, pyrimidine, pyrrole, indole, carbazole, furan,thiophene, imidazole, isoxazole, thiazole or their substituted or benzoderivatives) located either within the chain of the alkyl group or at aterminus; a substituted alicyclic group capable of complexing a metalion, C_(n) H_(2m) Y_(q) (n=1-30, 1≦q≦n, 1≦q≦n/2), where Y is O, S, Se,NH, N-R, N-Ar, PH, P-R and/or P-Ar or their acyl (including aminoacyl orpeptide) derivatives, and where R is saturated alkyl (C_(n) H_(2n),n=1-30), fluorinated saturated alkyl (C_(n) H_(q) F_(2n-q), n=1-30,0≦q≦2n), unsaturated alkyl (C_(n) H_(2m), n=1-30, 1≦m<n), fluorinatedunsaturated alkyl (C_(n) H_(q) F_(2m-q), n=1-30, 1≦m<n, 0≦q≦2m),alicyclic (C_(n) H_(2m), n=1-30, 1≦m≦n), fluorinated alicyclic (C_(n)H_(q) F_(2m-q), n=1-30, 1≦m<n, 0≦q≦2m), and where Ar is an aromaticmoiety (benzene, naphthalene, azulene, phenanthrene, anthracene,pyridine, quinoline, isoquinoline, purine, pyrimidine, pyrrole, indole,carbazole, furan, thiophene, imidazole, isoxazole, thiazole or theirsubstituted or benzo derivatives) located either within the chain of thealkyl group or at a terminus; or a modified or unmodified biomolecule (steroids, phospholipids, mono-, di- and triglycerides, mono- andpolysaccharides, nucleosides, and polypeptides), where Y is asubstituted aryl group (defined above), a charged group (CO₂ ⁻, SO₃ ⁻,PO₃ ²⁻, or ROPO₂ ²⁻), a silicon derivative (SiZ₃, where Z is alkyl,aryl, alkoxy, aryloxy, or halo), or a boron derivative (BZ₂, where Z isalkyl, aryl, alkoxy, aryloxy, or halo), or a biocompatible oligomer orpolymer (polyglycolic acid); or pharmaceutically acceptable saltsthereof.
 21. A bis-naphthalimide compound having the formula ##STR3##which is a mixture of stereoisomers wherein: R and R':saturatedstraight-chain or branched-chain alkyl, C_(n) H_(2n+1) (n=1-30);partially or totally fluorinated saturated straight-chain orbranched-chain alkyl, C_(n) H_(q) F_(2n-q+1) (n=1-30, 0≦q≦2n);unsaturated straight-chain or branched-chain alkyl, C_(n) H_(2m+1)(n=1-30, 1≦m<n); partially or totally fluorinated unsaturatedstraight-chain or branched-chain alkyl, C_(n) H_(q) F_(2m-q+1) (n=1-30,1≦m<n, 0≦q≦2m); alicyclic (monocyclic or polycyclic, fused-ring,bridged-ring or spirocyclic) alkyl, C_(n) H_(2m+1) (n=1-30, 1≦m≦n) withsaturated or unsaturated side-chains (branched or unbranched); partiallyor totally fluorinated alicyclic (monocyclic or polycyclic, fused-ring,bridged-ring or spirocyclic) alkyl, C_(n) H_(q) F_(2m-q+1) (n=1-30,1≦m<n, 0≦q≦2m) with saturated or unsaturated side-chains (branched orunbranched); aryl substituted branched, unbranched or alicyclic,saturated or unsaturated alkyl, ArC_(n) H_(2m+1) (n=1-30, 1≦m≦n), whereAr is an aromatic moiety (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole or their substituted or benzo derivatives) located eitherwithin the chain of the alkyl group or at a terminus; partially ortotally fluorinated aryl substituted branched, unbranched or alicyclic,saturated or unsaturated alkyl, ArC_(n) H_(q) F_(2m-q+1) (n=1-30, 1≦m≦n,0≦q≦2m), where Ar is an aromatic moiety (benzene, naphthalene, azulene,phenanthrene, anthracene, pyridine, quinoline, isoquinoline, purine,pyrimidine, pyrrole, indole, carbazole, furan, thiophene, imidazole,isoxazole, thiazole or their substituted or benzo derivatives) locatedeither within the chain of the alkyl group or at a terminus; substitutedsaturated straight-chain or branched-chain alkyl, C_(n) H_(2n-p+1) Y_(p)(n=1-30, 1≦p≦10) where Y is a substituted aryl group (benzene,naphthalene, azulene, phenanthrene, anthracene, pyridine, quinoline,isoquinoline, purine, pyrimidine, pyrrole, indole, carbazole, furan,thiophene, imidazole, isoxazole, thiazole or their substituted or benzoderivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, or ROPO₂ ²⁻), asilicon derivative (SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, orhalo), or a boron derivative (BZ₂, where Z is alkyl, aryl, alkoxy,aryloxy, or halo); substituted partially or totally fluorinatedstraight-chain or branched-chain alkyl, C_(n) H_(q) F_(2n-p-q+1) Y_(p)(n=1-30, 1≦p≦10 0≦q≦2n); substituted unsaturated straight-chain orbranched-chain alkyl, C_(n) H_(2m-p+1) Y_(p) (n=1-30, 1≦m<n, 1≦p≦10)where Y is a substituted aryl group (benzene, naphthalene, azulene,phenanthrene, anthracene, pyridine, quinoline, isoquinoline, purine,pyrimidine, pyrrole, indole, carbazole, furan, thiophene, imidazole,isoxazole, thiazole or their substituted or benzo derivatives), acharged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, or ROPO₂ ²⁻), a silicon derivative(SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, or halo), or a boronderivative (BZ₂, where Z is alkyl, aryl, alkoxy, aryloxy, or halo);substituted partially or totally fluorinated straight-chain orbranched-chain alkyl, C_(n) H_(q) F_(2m-p-q+1) Y_(p) (n=1-30, 1≦m<n,1≦p≦10, 0≦q≦2m); substituted alicyclic (monocyclic or polycyclic,fused-ring, bridged-ring or spirocyclic) alkyl, C_(n) H_(2m+1) (n=1-30,1≦m≦n) with saturated or unsaturated side-chains (branched orunbranched) where Y is a substituted aryl group (benzene, naphthalene,azulene, phenanthrene, anthracene, pyridine, quinoline, isoquinoline,purine, pyrimidine, pyrrole, indole, carbazole, furan, thiophene,imidazole, isoxazole, thiazole or their substituted or benzoderivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, or ROPO₂ ²⁻), asilicon derivative (SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, orhalo), or a boron derivative (BZ₂, where Z is alkyl, aryl, alkoxy,aryloxy, or halo); substituted partially or totally fluorinatedalicyclic (monocyclic or polycyclic, fused-ring, bridged-ring orspirocyclic) alkyl, C_(n) H_(q) F_(2m-p-q+1) Y_(p) (n=1-30, 1≦m<n,1≦p≦10, 0≦q≦2m) with saturated or unsaturated side-chains (branched orunbranched) where Y is a substituted aryl group (benzene, naphthalene,azulene, phenanthrene, anthracene, pyridine, quinoline, isoquinoline,purine, pyrimidine, pyrrole, indole, carbazole, furan, thiophene,imidazole, isoxazole, thiazole or their substituted or benzoderivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, or ROPO₂ ²⁻), asilicon derivative (SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, orhalo), or a boron derivative (BZ₂, where Z is alkyl, aryl, alkoxy,aryloxy, or halo); substituted aryl substituted branched, unbranched oralicyclic, saturated or unsaturated alkyl, ArC_(n) H_(2m-p+1) Y_(p)(n=1-30, 1≦m≦n, 1≦p≦10), where Ar is an aromatic moiety (benzene,naphthalene, azulene, phenanthrene, anthracene, pyridine, quinoline,isoquinoline, purine, pyrimidine, pyrrole, indole, carbazole, furan,thiophene, imidazole, isoxazole, thiazole or their substituted or benzoderivatives) located either within the chain of the alkyl group or at aterminus, and where Y is a substituted aryl group (defined above), acharged group (CO₂ ⁻, SO₃ ⁻, , PO₃ ²⁻, or ROPO₂ ²⁻), a siliconderivative (SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, or halo), ora boron derivative (BZ₂, where Z is alkyl, aryl, alkoxy, aryloxy, orhalo); substituted partially or totally fluorinated aryl substitutedbranched, unbranched or alicyclic, saturated or unsaturated alkyl,ArC_(n) H_(q) F_(2m-p-q+1) Y_(p) (n=1-30, 1≦m≦n, 1≦p≦10, 0≦q≦2m), whereAr is an aromatic moiety (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole or their substituted or benzo derivatives) located eitherwithin the chain of the alkyl group or at a terminus, and where Y is asubstituted aryl group (defined above), a charged group (CO₂ ⁻, SO₃ ⁻,PO₃ ²⁻, or ROPO₂ ²⁻), a silicon derivative (SiZ₃, where Z is alkyl,aryl, alkoxy, aryloxy, or halo), or a boron derivative (BZ₂, where Z isalkyl, aryl, alkoxy, aryloxy, or halo); a straight-chain orbranched-chain group capable of complexing a metal ion, C_(n) H_(2m+1)Y_(q) (n=1-30, 1≦q≦n, 1≦q≦n/2), where Y is O, S, Se, NH, N-R, N-Ar, PH,P-R and/or P-Ar or their acyl (including aminoacyl or peptide)derivatives, and where R is saturated alkyl (C_(n) H_(2n+1), n=1-30),fluorinated saturated alkyl (C_(n) H_(q) F_(2n-q+1), n=1-30, 0≦q≦2n),unsaturated alkyl (C_(n) H_(2m+1), n=1-30, 1≦m<n), fluorinatedunsaturated alkyl (C_(n) H_(q) F_(2m-q+1), n=1-30, 1≦m<n, 0≦q≦2m),alicyclic (C_(n) H_(2m+1), n=1-30, 1≦m≦n), fluorinated alicyclic (C_(n)H_(q) F_(2m-q+1), n=1-30, 1≦m<n, 0≦q≦2m), and where Ar is an aromaticmoiety (benzene, naphthalene, azulene, phenanthrene, anthracene,pyridine, quinoline, isoquinoline, purine, pyrimidine, pyrrole, indole,carbazole, furan, thiophene, imidazole, isoxazole, thiazole or theirsubstituted or benzo derivatives) located either within the chain of thealkyl group or at a terminus; a substituted alicyclic group capable ofcomplexing a metal ion, C_(n) H_(2m+1) Y_(q) (n=1-30, 1≦q≦n, 1≦q≦n/2),where Y is O, S, Se, NH, N-R, N-Ar, PH, P-R and/or P-Ar or their acyl(including aminoacyl or peptide) derivatives, and where R is saturatedalkyl (C_(n) H_(2n+1), n=1-30), fluorinated saturated alkyl (C_(n) H_(q)F_(2n-q+1), n=1-30, 0≦q≦2n), unsaturated alkyl (C_(n) H_(2m+1), n=1-30,1≦m<n), fluorinated unsaturated alkyl (C_(n) H_(q) F_(2m-q+1), n=1-30,1≦m<n, 0≦q≦2m), alicyclic (C_(n) H_(2m+1), n=1-30, 1≦m≦n), fluorinatedalicyclic (C_(n) H_(q) F_(2m-q+1), n=1-30, 1≦m<n, 0≦q≦2m), and where Aris an aromatic moiety (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole or their substituted or benzo derivatives) located eitherwithin the chain of the alkyl group or at a terminus; or a modified orunmodified biomolecule (steroids, phospholipids, mono-, di- andtriglycerides, mono- and polysaccharides, nucleosides, andpolypeptides), where Y is a substituted aryl group (defined above), acharged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, and ROPO₂ ²⁻), a silicon derivative(SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, or halo), or a boronderivative (BZ₂, where Z is alkyl, aryl, alkoxy, aryloxy, or halo), or abiocompatible oligomer or polymer (polyglycolic acid);X: halogen (F, Cl,Br, I); sulfonate ester (alkanesulfonates, partially or totallyfluorinated alkanesulfonates, arenesulfonates, or partially or totallyfluorinated arenesulfonates); or a nitrogen leaving group (diazoniumion);Q: saturated straight-chain or branched-chain alkyl, C_(n) H_(2n)(n=1-30); partially or totally fluorinated saturated straight-chain orbranched-chain alkyl, C_(n) H_(q) F_(2n-q) (n=1-30, 0≦q≦2n); unsaturatedstraight-chain or branched-chain alkyl, C_(n) H_(2m) (n=1-30, 1≦m<n);partially or totally fluorinated unsaturated straight-chain orbranched-chain alkyl, C_(n) H_(q) F_(2m-q) (n=1-30, 1≦m<n, 0≦q≦2m);alicyclic (monocyclic or polycyclic, fused-ring, bridged-ring orspirocyclic) alkyl, C_(n) H_(2m) (n=1-30, 1≦m≦n) with saturated orunsaturated side-chains (branched or unbranched); partially or totallyfluorinated alicyclic (monocyclic or polycyclic, fused-ring,bridged-ring or spirocyclic) alkyl, C_(n) H_(q) F_(2m-q) (n=1-30, 1≦m<n,0≦q≦2m) with saturated or unsaturated side-chains (branched orunbranched); aryl substituted branched, unbranched or alicyclic,saturated or unsaturated alkyl, ArC_(n) H_(2m) (n=1-30, 1≦m≦n), where Aris an aromatic moiety (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole or their substituted or benzo derivatives) located eitherwithin the chain of the alkyl group or at a terminus: partially ortotally fluorinated aryl substituted branched, unbranched or alicyclic,saturated or unsaturated alkyl, ArC_(n) H_(q) F_(2m-q) (n=1-30, 1≦m≦n,0≦q≦2m), where Ar is an aromatic moiety (benzene, naphthalene, azulene,phenanthrene, anthracene, pyridine, quinoline, isoquinoline, purine,pyrimidine, pyrrole, indole, carbazole, furan, thiophene, imidazole,isoxazole, thiazole or their substituted or benzo derivatives) locatedeither within the chain of the alkyl group or at a terminus; substitutedsaturated straight-chain or branched-chain alkyl, C_(n) H_(2n-p) Y_(p)(n=1-30, 1≦p≦10) where Y is a substituted aryl group (benzene,naphthalene, azulene, phenanthrene, anthracene, pyridine, quinoline,isoquinoline, purine, pyrimidine, pyrrole, indole, carbazole, furan,thiophene, imidazole, isoxazole, thiazole or their substituted or benzoderivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, or ROPO₂ ²⁻), asilicon derivative (SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, orhalo), or a boron derivative (BZ₂, where Z is alkyl, aryl, alkoxy,aryloxy, or halo); substituted partially or totally fluorinatedstraight-chain or branched-chain alkyl, C_(n) H_(q) F_(2n-p-q) Y_(p)(n=1-30, 1≦p≦10 0≦q≦2n); substituted unsaturated straight-chain orbranched-chain alkyl, C_(n) H_(2m-p) Y_(p) (n=1-30, 1≦m<n, 1≦p≦10) whereY is a substituted aryl group (benzene, naphthalene, azulene,phenanthrene, anthracene, pyridine, quinoline, isoquinoline, purine,pyrimidine, pyrrole, indole, carbazole, furan, thiophene, imidazole,isoxazole, thiazole or their substituted or benzo derivatives), acharged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, or ROPO₂ ²⁻), a silicon derivative(SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, or halo), or a boronderivative (BZ₂, where Z is alkyl, aryl, alkoxy, aryloxy, or halo);substituted partially or totally fluorinated straight-chain orbranched-chain alkyl, C_(n) H_(q) F_(2m-p-q) Y_(p) (n=1-30, 1≦m<n,1≦p≦10, 0≦q≦2m); substituted alicyclic (monocyclic or polycyclic,fused-ring, bridged-ring or spirocyclic) alkyl, C_(n) H_(2m) (n=1-30,1≦m≦n) with saturated or unsaturated side-chains (branched orunbranched) where Y is a substituted aryl group (benzene, naphthalene,azulene, phenanthrene, anthracene, pyridine, quinoline, isoquinoline,purine, pyrimidine, pyrrole, indole, carbazole, furan, thiophene,imidazole, isoxazole, thiazole or their substituted or benzoderivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, or ROPO₂ ²⁻), asilicon derivative (SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, orhalo), or a boron derivative (BZ₂, where Z is alkyl, aryl, alkoxy,aryloxy, or halo): substituted partially or totally fluorinatedalicyclic (monocyclic or polycyclic, fused-ring, bridged-ring orspirocyclic) alkyl, C_(n) H_(q) F_(2m-p-q) Y_(p) (n=1-30, 1≦m<n, 1≦p≦10,0≦q≦2m) with saturated or unsaturated side-chains (branched orunbranched) where Y is a substituted aryl group (benzene, naphthalene,azulene, phenanthrene, anthracene, pyridine, quinoline, isoquinoline,purine, pyrimidine, pyrrole, indole, carbazole, furan, thiophene,imidazole, isoxazole, thiazole or their substituted or benzoderivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, or ROPO₂ ²⁻), asilicon derivative (SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, orhalo), or a boron derivative (BZ₂, where Z is alkyl, aryl, alkoxy,aryloxy, or halo); substituted aryl substituted branched, unbranched oralicyclic, saturated or unsaturated alkyl, ArC_(n) H_(2m-p) Y_(p)(n=1-30, 1≦m≦n, 1≦p≦10), where Ar is an aromatic moiety (benzene,naphthalene, azulene, phenanthrene, anthracene, pyridine, quinoline,isoquinoline, purine, pyrimidine, pyrrole, indole, carbazole, furan,thiophene, imidazole, isoxazole, thiazole or their substituted or benzoderivatives) located either within the chain of the alkyl group or at aterminus, and where Y is a substituted aryl group (defined above), acharged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, or ROPO₂ ²⁻), a silicon derivative(SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, or halo), or a boronderivative (BZ₂, where Z is alkyl, aryl, alkoxy, aryloxy, or halo);substituted partially or totally fluorinated aryl substituted branched,unbranched or alicyclic, saturated or unsaturated alkyl, ArC_(n) H_(q)F_(2m-p-q) Y_(p) (n=1-30, 1≦m≦n, 1≦p≦10, 0≦q≦2m), where Ar is anaromatic moiety (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole or their substituted or benzo derivatives) located eitherwithin the chain of the alkyl group or at a terminus, and where Y is asubstituted aryl group (defined above), a charged group (CO₂ ⁻, SO₃ ⁻,PO₃ ²⁻, or ROPO₂ ²⁻), a silicon derivative (SiZ₃, where Z is alkyl,aryl, alkoxy, aryloxy, or halo), or a boron derivative (BZ₂, where Z isalkyl, aryl, alkoxy, aryloxy, or halo); a straight-chain orbranched-chain group capable of complexing a metal ion, C_(n) H_(2m)Y_(q) (n=1-30, 1≦q≦n, 1≦q≦n/2), where Y is O, S, Se, NH, N-R, N-Ar, PH,P-R and/or P-Ar or their acyl (including aminoacyl or peptide)derivatives, and where R is saturated alkyl (C_(n) H_(2n), n=1-30),fluorinated saturated alkyl (C_(n) H_(q) F_(2n-q), n=1-30, 0≦q≦2n),unsaturated alkyl (C_(n) H_(2m), n=1-30, 1≦m<n), fluorinated unsaturatedalkyl (C_(n) H_(q) F_(2m-q), n=1-30, 1≦m<n, 0≦q≦2m), alicyclic (C_(n)H_(2m), n=1-30, 1≦m≦n), fluorinated alicyclic (C_(n) H_(q) F_(2m-q),n=1-30, 1≦m≦n, 0≦q≦2m), and where Ar is an aromatic moiety (benzene,naphthalene, azulene, phenanthrene, anthracene, pyridine, quinoline,isoquinoline, purine, pyrimidine, pyrrole, indole, carbazole, furan,thiophene, imidazole, isoxazole, thiazole or their substituted and benzoderivatives) located either within the chain of the alkyl group or at aterminus; a substituted alicyclic group capable of complexing a metalion, C_(n) H_(2m) Y_(q) (n=1-30, 1≦q≦n, 1≦q≦n/2), where Y is O, S, Se,NH, N-R, N-Ar, PH, P-R and/or P-Ar or their acyl (including aminoacyl orpeptide) derivatives, and where R is saturated alkyl (C_(n) H_(2n),n=1-30), fluorinated saturated alkyl (C_(n) H_(q) F_(2n-q), n=1-30,0≦q≦2n), unsaturated alkyl (C_(n) H_(2m), n=1-30, 1≦m<n), fluorinatedunsaturated alkyl (C_(n) H_(q) F_(2m-q), n=1-30, 1≦m<n, 0≦q≦2m),alicyclic (C_(n) H_(2m), n=1-30, 1≦m≦n), fluorinated alicyclic (C_(n)H_(q) F_(2m-q), n=1-30, 1≦m<n, 0≦q≦2m), and where Ar is an aromaticmoiety (benzene, naphthalene, azulene, phenanthrene, anthracene,pyridine, quinoline, isoquinoline, purine, pyrimidine, pyrrole, indole,carbazole, furan, thiophene, imidazole, isoxazole, thiazole or theirsubstituted or benzo derivatives) located either within the chain of thealkyl group or at a terminus; or a modified or unmodified biomolecule (steroids, phospholipids, mono-, di- and triglycerides, mono- orpolysaccharides, nucleosides, or polypeptides), where Y is a substitutedaryl group (defined above), a charged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, andROPO₂ ²⁻), a silicon derivative (SiZ₃, where Z is alkyl, aryl, alkoxy,aryloxy, or halo), or a boron derivative (BZ₂, where Z is alkyl, aryl,alkoxy, aryloxy, or halo), or a biocompatible oligomer or polymer(polyglycolic acid); or pharmaceutically acceptable salts thereof.
 22. Abis-naphthalimide compound having the formula ##STR4## which is amixture of stereoisomers wherein: R and R':saturated straight-chain orbranched-chain alkyl, C_(n) H_(2n+1) (n=1-30); partially or totallyfluorinated saturated straight-chain or branched-chain alkyl, C_(n)H_(q) F_(2n-q+1) (n=1-30, 0≦q≦2n); unsaturated straight-chain orbranched-chain alkyl, C_(n) H_(2m+1) (n=1-30, 1≦m<n); partially ortotally fluorinated unsaturated straight-chain or branched-chain alkyl,C_(n) H_(q) F_(2m-q+1) (n=1-30, 1≦m<n, 0≦q≦2m); alicyclic (monocyclic orpolycyclic, fused-ring, bridged-ring or spirocyclic) alkyl, C_(n)H_(2m+1) (n=1-30, 1≦m≦n) with saturated or unsaturated side-chains(branched or unbranched); partially or totally fluorinated alicyclic(monocyclic or polycyclic, fused-ring, bridged-ring or spirocyclic)alkyl, C_(n) H_(q) F_(2m-q+1) (n=1-30, 1≦m<n, 0≦q≦2m) with saturated orunsaturated side-chains (branched or unbranched); aryl substitutedbranched, unbranched or alicyclic, saturated or unsaturated alkyl,aromatic moiety (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole or their substituted or benzo derivatives) located eitherwithin the chain of the alkyl group or at a terminus; partially ortotally fluorinated aryl substituted branched, unbranched or alicyclic,saturated or unsaturated alkyl, ArC_(n) H_(q) F_(2m-q+1) (n=1-30, 1≦m≦n,0≦q≦2m), where Ar is an aromatic moiety (benzene, naphthalene, azulene,phenanthrene, anthracene, pyridine, quinoline, isoquinoline, purine,pyrimidine, pyrrole, indole, carbazole, furan, thiophene, imidazole,isoxazole, thiazole or their substituted or benzo derivatives) locatedeither within the chain of the alkyl group or at a terminus; substitutedsaturated straight-chain or branched-chain alkyl, C_(n) H_(2n-p+1) Y_(p)(n=1-30, 1≦p≦10) where Y is a substituted aryl group (benzene,naphthalene, azulene, phenanthrene, anthracene, pyridine, quinoline,isoquinoline, purine, pyrimidine, pyrrole, indole, carbazole, furan,thiophene, imidazole, isoxazole, thiazole or their substituted or benzoderivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, or ROPO₂ ²⁻), asilicon derivative (SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, orhalo), or a boron derivative (BZ₂, where Z is alkyl, aryl, alkoxy,aryloxy, or halo); substituted partially or totally fluorinatedstraight-chain or branched-chain alkyl, C_(n) H_(q) F_(2n-p-q+1) Y_(p)(n=1-30, 1≦p≦10 0≦q≦2n); substituted unsaturated straight-chain orbranched-chain alkyl, C_(n) H_(2m-p+1) Y_(p) (n=1-30, 1≦m<n, 1≦p≦10)where Y is a substituted aryl group (benzene, naphthalene, azulene,phenanthrene, anthracene, pyridine, quinoline, isoquinoline, purine,pyrimidine, pyrrole, indole, carbazole, furan, thiophene, imidazole,isoxazole, thiazole or their substituted or benzo derivatives), acharged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, or ROPO₂ ²⁻), a silicon derivative(SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, or halo), or a boronderivative (BZ₂, where Z is alkyl, aryl, alkoxy, aryloxy, or halo):substituted partially or totally fluorinated straight-chain orbranched-chain alkyl, C_(n) H_(q) F_(2m-p-q+1) Y_(p) (n=1-30, 1≦m<n,1≦p≦10, 0≦q≦2m): substituted alicyclic (monocyclic or polycyclic,fused-ring, bridged-ring or spirocyclic) alkyl, C_(n) H_(2m+1) (n=1-30,1≦m≦n) with saturated or unsaturated side-chains (branched orunbranched) where Y is a substituted aryl group (benzene, naphthalene,azulene, phenanthrene, anthracene, pyridine, quinoline, isoquinoline,purine, pyrimidine, pyrrole, indole, carbazole, furan, thiophene,imidazole, isoxazole, thiazole or their substituted or benzoderivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, or ROPO₂ ²⁻), asilicon derivative (SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, orhalo), or a boron derivative (BZ₂, where Z is alkyl, aryl, alkoxy,aryloxy, or halo); substituted partially or totally fluorinatedalicyclic (monocyclic or polycyclic, fused-ring, bridged-ring orspirocyclic) alkyl, C_(n) H_(q) F_(2m-p-q+1) Y_(p) (n=1-30, 1≦m<n,1≦p≦10, 0≦q≦2m) with saturated or unsaturated side-chains (branched orunbranched) where Y is a substituted aryl group (benzene, naphthalene,azulene, phenanthrene, anthracene, pyridine, quinoline, isoquinoline,purine, pyrimidine, pyrrole, indole, carbazole, furan, thiophene,imidazole, isoxazole, thiazole or their substituted or benzoderivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, and ROPO₂ ²⁻), asilicon derivative (SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, orhalo), or a boron derivative (BZ₂, where Z is alkyl, aryl, alkoxy,aryloxy, or halo); substituted aryl substituted branched, unbranched oralicyclic, saturated or unsaturated alkyl, naphthalene, azulene,phenanthrene, anthracene, pyridine, quinoline, isoquinoline, purine,pyrimidine, pyrrole, indole, carbazole, furan, thiophene, imidazole,isoxazole, thiazole or their substituted or benzo derivatives) locatedeither within the chain of the alkyl group or at a terminus, and where Yis a substituted aryl group (defined above), a charged group (CO₂ ⁻, SO₃⁻, PO₃ ²⁻, or ROPO₂ ²⁻), a silicon derivative (SiZ₃, where Z is alkyl,aryl, alkoxy, aryloxy, or halo), or a boron derivative (BZ₂, where Z isalkyl, aryl, alkoxy, aryloxy, or halo); substituted partially or totallyfluorinated aryl substituted branched, unbranched or alicyclic,saturated or unsaturated alkyl, ArC_(n) H_(q) F_(2m-p-q+1) Y_(p)(n=1-30, 1≦m≦n, 1≦p≦10, 0≦q≦2m), where Ar is an aromatic moiety(benzene, naphthalene, azulene, phenanthrene, anthracene, pyridine,quinoline, isoquinoline, purine, pyrimidine, pyrrole, indole, carbazole,furan, thiophene, imidazole, isoxazole, thiazole or their substituted orbenzo derivatives) located either within the chain of the alkyl group orat a terminus, and where Y is a substituted aryl group (defined above),a charged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, and ROPO₂ ²⁻), a siliconderivative (SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, or halo), ora boron derivative (BZ₂, where Z is alkyl, aryl, alkoxy, aryloxy, orhalo); a straight-chain or branched-chain group capable of complexing ametal ion, C_(n) H_(2m+1) Y_(q) (n=1-30, 1≦q≦n, 1≦q≦n/2), where Y is O,S, Se, NH, N-R, N-Ar, PH, P-R and/or P-Ar and their acyl (includingaminoacyl or peptide) derivatives, and where R is saturated alkyl (C_(n)H_(2n+1), n=1-30), fluorinated saturated alkyl (C_(n) H_(q) F_(2n-q+1),n=1-30, 0≦q≦2n), unsaturated alkyl (C_(n) H_(2m+1), n=1-30, 1≦m<n),fluorinated unsaturated alkyl (C_(n) H_(q) F_(2m-q+1), n=1-30, 1≦m<n,0≦q≦2m), alicyclic (C_(n) H_(2m+1), n=1-30, 1≦m≦n), fluorinatedalicyclic (C_(n) H_(q) F_(2m-q+1), n=1-30, 1≦m<n, 0≦q≦2m), and where Aris an aromatic moiety (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole or their substituted or benzo derivatives) located eitherwithin the chain of the alkyl group or at a terminus; a substitutedalicyclic group capable of complexing a metal ion, C_(n) H_(2m+1) Y_(q)(n=1-30, 1≦q≦n, 1≦q≦n/2), where Y is O, S, Se, NH, N-R, N-Ar, PH, P-Rand/or P-Ar and their acyl (including aminoacyl or peptide) derivatives,and where R is saturated alkyl (C_(n) H_(2n+1), n=1-30), fluorinatedsaturated alkyl (C_(n) H_(q) F_(2n-q+1), n=1-30, 0≦q≦2n), unsaturatedalkyl (C_(n) H_(2m+1), n=1-30, 1≦m≦n), fluorinated unsaturated alkyl(C_(n) H_(q) F_(2m-q+1), n=1-30, 1≦m<n, 0≦q≦2m), alicyclic (C_(n)H_(2m+1), n=1-30, 1≦m≦n), fluorinated alicyclic (C_(n) H_(q) F_(2m-q+1),n=1-30, 1≦m<n, 0≦q≦2m), and where Ar is an aromatic moiety (benzene,naphthalene, azulene, phenanthrene, anthracene, pyridine, quinoline,isoquinoline, purine, pyrimidine, pyrrole, indole, carbazole, furan,thiophene, imidazole, isoxazole, thiazole or their substituted or benzoderivatives) located either within the chain of the alkyl group or at aterminus; or a modified or unmodified biomolecule (steroids,phospholipids, mono-, di- and triglycerides, mono- and polysaccharides,nucleosides, and polypeptides), where Y is a substituted aryl group(defined above), a charged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, and ROPO₂ ²⁻), asilicon derivative (SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, orhalo), or a boron derivative (BZ₂, where Z is alkyl, aryl, alkoxy,aryloxy, or halo), or a biocompatible oligomer or polymer (polyglycolicacid);X: halogen (F, Cl, Br, I); sulfonate ester (alkanesulfonates,partially or totally fluorinated alkanesulfonates, arenesulfonates, orpartially or totally fluorinated arenesulfonates); or a nitrogen leavinggroup (diazonium ion);Q: saturated straight-chain or branched-chainalkyl, C_(n) H_(2n) (n=1-30); partially or totally fluorinated saturatedstraight-chain or branched-chain alkyl, C_(n) H_(q) F_(2n-q) (n=1-30,0≦q≦2n); unsaturated straight-chain or branched-chain alkyl, C_(n)H_(2m) (n=1-30, 1≦m<n); partially or totally fluorinated unsaturatedstraight-chain or branched-chain alkyl, C_(n) H_(q) F_(2m-q) (n=1-30,1≦m<n, 0≦q≦2m); alicyclic (monocyclic or polycyclic, fused-ring,bridged-ring or spirocyclic) alkyl, C_(n) H_(2m) (n=1-30, 1≦m≦n) withsaturated or unsaturated side-chains (branched or unbranched); partiallyor totally fluorinated alicyclic (monocyclic or polycyclic, fused-ring,bridged-ring or spirocyclic) alkyl, C_(n) H_(q) F_(2m-q) (n=1-30, 1≦m<n,0≦q≦2m) with saturated or unsaturated side-chains (branched orunbranched); aryl substituted branched, unbranched or alicyclic,saturated or unsaturated alkyl, ArC_(n) H_(2m) (n=1-30, 1≦m≦n), where Aris an aromatic moiety (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole or their substituted or benzo derivatives) located eitherwithin the chain of the alkyl group or at a terminus; partially ortotally fluorinated aryl substituted branched, unbranched or alicyclic,saturated or unsaturated alkyl, ArC_(n) H_(q) F_(2m-q) (n=1-30, 1≦m≦n,0≦q≦2m), where Ar is an aromatic moiety (benzene, naphthalene, azulene,phenanthrene, anthracene, pyridine, quinoline, isoquinoline, purine,pyrimidine, pyrrole, indole, carbazole, furan, thiophene, imidazole,isoxazole, thiazole or their substituted or benzo derivatives) locatedeither within the chain of the alkyl group or at a terminus; substitutedsaturated straight-chain or branched-chain alkyl, C_(n) H_(2n-p) Y_(p)(n=1-30, 1≦p≦10) where Y is a substituted aryl group (benzene,naphthalene, azulene, phenanthrene, anthracene, pyridine, quinoline,isoquinoline, purine, pyrimidine, pyrrole, indole, carbazole, furan,thiophene, imidazole, isoxazole, thiazole or their substituted or benzoderivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, and ROPO₂ ²⁻), asilicon derivative (SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, orhalo), or a boron derivative (BZ₂, where Z is alkyl, aryl, alkoxy,aryloxy, or halo): substituted partially or totally fluorinatedstraight-chain or branched-chain alkyl, C_(n) H_(q) F_(2n-p-q) Y_(p)(n=1-30, 1≦p≦10 0≦q≦2n); substituted unsaturated straight-chain orbranched-chain alkyl, C_(n) H_(2m-p) Y_(p) (n=1-30, 1≦m<n, 1≦p≦10) whereY is a substituted aryl group (benzene, naphthalene, azulene,phenanthrene, anthracene, pyridine, quinoline, isoquinoline, purine,pyrimidine, pyrrole, indole, carbazole, furan, thiophene, imidazole,isoxazole, thiazole or their substituted or benzo derivatives), acharged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, and ROPO₂ ²⁻), a silicon derivative(SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, or halo), or a boronderivative (BZ₂, where Z is alkyl, aryl, alkoxy, aryloxy, or halo);substituted partially or totally fluorinated straight-chain orbranched-chain alkyl, C_(n) H_(q) F_(2m-p-q) Y_(p) (n=1-30, 1≦m<n,1≦p≦10, 0≦q≦2m); substituted alicyclic (monocyclic or polycyclic,fused-ring, bridged-ring or spirocyclic) alkyl, C_(n) H_(2m) (n=1-30,1≦m≦n) with saturated or unsaturated side-chains (branched orunbranched) where Y is a substituted aryl group (benzene, naphthalene,azulene, phenanthrene, anthracene, pyridine, quinoline, isoquinoline,purine, pyrimidine, pyrrole, indole, carbazole, furan, thiophene,imidazole, isoxazole, thiazole or their substituted or benzoderivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, and ROPO₂ ²⁻), asilicon derivative (SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, orhalo), or a boron derivative (BZ₂, where Z is alkyl, aryl, alkoxy,aryloxy, or halo); substituted partially or totally fluorinatedalicyclic (monocyclic or polycyclic, fused-ring, bridged-ring orspirocyclic) alkyl, C_(n) H_(q) F_(2m-p-q) Y_(p) (n=1-30, 1≦m<n, 1≦p≦10,0≦q≦2m) with saturated or unsaturated side-chains (branched orunbranched) where Y is a substituted aryl group (benzene, naphthalene,azulene, phenanthrene, anthracene, pyridine, quinoline, isoquinoline,purine, pyrimidine, pyrrole, indole, carbazole, furan, thiophene,imidazole, isoxazole, thiazole or their substituted or benzoderivatives), a charged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, and ROPO₂ ²⁻), asilicon derivative (SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, orhalo), or a boron derivative (BZ₂, where Z is alkyl, aryl, alkoxy,aryloxy, or halo); substituted aryl substituted branched, unbranched oralicyclic, saturated or unsaturated alkyl, ArC_(n) H_(2m-p) Y_(p)(n=1-30, 1≦m≦n, 1≦p≦10), where Ar is an aromatic moiety (benzene,naphthalene, azulene, phenanthrene, anthracene, pyridine, quinoline,isoquinoline, purine, pyrimidine, pyrrole, indole, carbazole, furan,thiophene, imidazole, isoxazole, thiazole or their substituted or benzoderivatives) located either within the chain of the alkyl group or at aterminus, and where Y is a substituted aryl group (defined above), acharged group (CO₂ ⁻, SO₃ ⁻, PO₃ ²⁻, and ROPO₂ ²⁻), a silicon derivative(SiZ₃, where Z is alkyl, aryl, alkoxy, aryloxy, or halo), or a boronderivative (BZ₂, where Z is alkyl, aryl, alkoxy, aryloxy, or halo);substituted partially or totally fluorinated aryl substituted branched,unbranched or alicyclic, saturated or unsaturated alkyl, ArC_(n) H_(q)F_(2m-p-q) Y_(p) (n=1-30, 1≦m≦n, 1≦p≦10, 0≦q≦2m), where Ar is anaromatic moiety (benzene, naphthalene, azulene, phenanthrene,anthracene, pyridine, quinoline, isoquinoline, purine, pyrimidine,pyrrole, indole, carbazole, furan, thiophene, imidazole, isoxazole,thiazole or their substituted or benzo derivatives) located eitherwithin the chain of the alkyl group or at a terminus, and where Y is asubstituted aryl group (defined above), a charged group (CO₂ ⁻, SO₃ ⁻,PO₃ ²⁻, and ROPO₂ ²⁻), a silicon derivative (SiZ₃, where Z is alkyl,aryl, alkoxy, aryloxy, or halo), or a boron derivative (BZ₂, where Z isalkyl, aryl, alkoxy, aryloxy, or halo); a straight-chain orbranched-chain group capable of complexing a metal ion, C_(n) H_(2m)Y_(q) (n=1-30, 1≦q≦n, 1≦q≦n/2), where Y is O, S, Se, NH, N-R, N-Ar, PH,P-R and/or P-Ar or their acyl (including aminoacyl and peptide)derivatives, and where R is saturated alkyl (C_(n) H_(2n), n=1-30),fluorinated saturated alkyl (C_(n) H_(q) F_(2n-q), n=1-30, 0≦q≦2n),unsaturated alkyl (C_(n) H_(2m), n=1-30, 1≦m<n), fluorinated unsaturatedalkyl (C_(n) H_(q) F_(2m-q), n=1-30, 1≦m<n, 0≦q≦2m), alicyclic (C_(n)H_(2m), n=1-30, 1≦m≦n), fluorinated alicyclic (C_(n) H_(q) F_(2m-q),n=1-30, 1≦m<n, 0≦q≦2m), and where Ar is an aromatic moiety (benzene,naphthalene, azulene, phenanthrene, anthracene, pyridine, quinoline,isoquinoline, purine, pyrimidine, pyrrole, indole, carbazole, furan,thiophene, imidazole, isoxazole, thiazole or their substituted or benzoderivatives) located either within the chain of the alkyl group or at aterminus; a substituted alicyclic group capable of complexing a metalion, C_(n) H_(2m) Y_(q) (n=1-30, 1≦q≦n/2), where Y is O, S, Se, NH, N-R,N-Ar, PH, P-R and/or P-Ar and their acyl (including aminoacyl andpeptide) derivatives, and where R is saturated alkyl (C_(n) H_(2n),n=1-30), fluorinated saturated alkyl (C_(n) H_(q) F_(2n-q), n=1-30,0≦q≦2n), unsaturated alkyl (C_(n) H_(2m), n=1-30, 1≦m<n), fluorinatedunsaturated alkyl (C_(n) H_(q) F_(2m-q), n=1-30, 1≦m<n, 0≦q≦2m),alicyclic (C_(n) H_(2m), n=1-30, 1≦m≦n), fluorinated alicyclic (C_(n)H_(q) F_(2m-q), n=1-30, 1≦m<n, 0≦q≦2m), and where Ar is an aromaticmoiety (benzene, naphthalene, azulene, phenanthrene, anthracene,pyridine, quinoline, isoquinoline, purine, pyrimidine, pyrrole, indole,carbazole, furan, thiophene, imidazole, isoxazole, thiazole or theirsubstituted or benzo derivatives) located either within the chain of thealkyl group or at a terminus; or a modified or unmodified biomolecule(steroids, phospholipids, mono-, di- and triglycerides, mono- andpolysaccharides, nucleosides, and polypeptides), where Y is asubstituted aryl group (defined above), a charged group (CO₂ ⁻, SO₃ ⁻,PO₃ ²⁻, and ROPO₂ ²⁻), a silicon derivative (SiZ₃, where Z is alkyl,aryl, alkoxy, aryloxy, or halo), or a boron derivative (BZ₂, where Z isalkyl, aryl, alkoxy, aryloxy, or halo), or a biocompatible oligomer orpolymer (polyglycolic acid); or pharmaceutically acceptable saltsthereof.
 23. A pharmaceutical composition for eradicating a pathogenicbiological contaminant from body tissue, which comprises atherapeutically effective amount of a compound of claim 1, or apharmaceutically acceptable salt thereof, having been activated by asufficient amount of an activating agent, as an active ingredient, incombination with a pharmaceutically acceptable carrier for said activeingredient.
 24. The composition of claim 23 wherein said body tissue isbody fluid, packed red blood cell, packed white blood cell, cryoprecipitate from blood plasma, plasma protein, skin or cornea.
 25. Thecomposition of claim 24 wherein said body fluid is whole blood, bloodplasma, serum, fluid from plasmapheresis, bone marrow or plasmafibrinogen.
 26. The composition of claim 24 wherein said body fluid isalbumin, gamma globulin or semen.
 27. The composition of claim 23wherein said body tissue is platelet.
 28. The composition of claim 23wherein said pathogenic biological contaminant is virus, tumor cell,bacterium or parasite.
 29. A pharmaceutical composition for eradicatinga pathogenic biological contaminant from body tissue, which comprises atherapeutically effective amount of a compound of claim 11, or apharmaceutically acceptable salt thereof, having been activated by asufficient amount of an activating agent, as an active ingredient, incombination with a pharmaceutically acceptable carrier for said activeingredient.
 30. The composition of claim 29 wherein said body tissue isbody fluid, packed red blood cell, packed white blood cell, cryoprecipitate from blood plasma, plasma protein, skin or cornea.
 31. Thecomposition of claim 30 wherein said body fluid is whole blood, bloodplasma, serum, fluid from plasmapheresis, bone marrow or plasmafibrinogen.
 32. The composition of claim 30 wherein said body fluid isalbumin, gamma globulin or semen.
 33. The composition of claim 29wherein said body tissue is platelet.
 34. The composition of claim 29wherein said pathogenic biological contaminant is virus, tumor cell,bacterium or parasite.
 35. A composition for cross-linking protein,which comprises an effective amount of a compound of claim 11, or anacceptable salt thereof, having been activated by a sufficient amount ofan activating agent, as an active ingredient, in combination with anacceptable carrier for said active ingredient.